EP4526333A2 - Compositions et méthodes d'ingénierie de cellules - Google Patents

Compositions et méthodes d'ingénierie de cellules

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Publication number
EP4526333A2
EP4526333A2 EP23808207.7A EP23808207A EP4526333A2 EP 4526333 A2 EP4526333 A2 EP 4526333A2 EP 23808207 A EP23808207 A EP 23808207A EP 4526333 A2 EP4526333 A2 EP 4526333A2
Authority
EP
European Patent Office
Prior art keywords
cell
nucleic acid
certain embodiments
sequence
nuclease
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23808207.7A
Other languages
German (de)
English (en)
Inventor
Tanya Warnecke
Roland Baumgartner
John SCHIEL
Nicholas Eion Timmins
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Celyntra Therapeutics SA
Original Assignee
Celyntra Therapeutics SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Celyntra Therapeutics SA filed Critical Celyntra Therapeutics SA
Publication of EP4526333A2 publication Critical patent/EP4526333A2/fr
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • Chimeric antigen receptor (CAR) T cells are T cells that have been genetically engineered to produce an artificial T cell receptor for use in one or more therapies.
  • the receptors are “chimeric” because they combine both antigen-binding and T cell activating functions into a single receptor.
  • T cells comprising engineered CARs, for therapeutic purposes, also need to distinguish “self” from “non-self”, as recognition “host” cells as “foreign” can elicit an immune response, i.e., “Graft versus Host” (GvH), resulting in the therapy demonstrating a negative and/or harmful effect on the recipient.
  • GvH raft versus Host
  • Figure 1A shows a schematic representation showing the structure of an exemplary single guide Type V-A CRISPR system.
  • Figure 1B is a schematic representation showing the structure of an exemplary dual guide Type V-A CRISPR system.
  • Figures 2A-C show a series of schematic representation showing incorporation of a protecting group (e.g., a protective nucleotide sequence or a chemical modification) (Figure 2A), a donor template-recruiting sequence ( Figure 2B), and an editing enhancer (Figure 2C) into a Type V-A CRISPR-Cas system.
  • a protecting group e.g., a protective nucleotide sequence or a chemical modification
  • Figure 2B e.g., a donor template-recruiting sequence
  • an editing enhancer Figure 2C
  • Figure 3 shows resulting populations after engineering of a starting, target cell with a composition comprising a plurality of polynucleotides encoding polypeptides comprising CARs or portions thereof.
  • Figure 4 shows a schematic of dual CARs linked via a polypeptide linker.
  • Figure 5 shows a schematic of surface expressed polypeptides comprising CARs or portions thereof and secreted polypeptides comprising CARs or portions thereof.
  • Figure 6 show a schematic of a Type V-A nucleic acid guide nuclease comprising a dual guide nucleic acid.
  • Figure 7 shows delivery of one or more components of a genome editing system: (A) illustrates transfer of the components to the nucleus; (B) shows transfer of the components wherein at least some of the components are bound together, to the nucleus.
  • Figures 8A-B show an exemplary method for engineering genomes to comprise one or more genomic modifications either sequentially, simultaneously, or both: ( Figure 8A) without intervening growth and/or differentiation steps; ( Figure 8B) with optional intervening growth and/or differentiation steps.
  • CARs engineered chimeric antigen receptors
  • Cells comprising polynucleotides encoding polypeptides and/or polypeptides comprising CARs or portions thereof D.
  • Cell populations comprising CARs E.
  • Engineered, non-naturally occurring dual guide CRISPR-cas systems A. Cas proteins
  • Guide nucleic acids C. gNA modifications
  • Composition and methods for targeting, editing, and/or modifying genomic DNA A. Ribonucleotide protein (RNP) delivery and “cas RNA” delivery
  • CRISPR expression systems C. Donor templates D. Efficiency and specificity E. Multiplex F. Genomic safe harbors V. Therapeutic uses A. Gene therapies VI. Kits VII.
  • compositions, methods, and/or kits comprising polynucleotides encoding polypeptides comprising CARs or portions thereof and/or polypeptides comprising CARs or portions thereof. Further, provided herein are compositions, methods, and/or kits comprising a cell comprising polynucleotides encoding and/or polypeptides comprising CARs or portions thereof and/or progeny of such cells, e.g., cells comprising polypeptides comprising CARs or portions thereof.
  • compositions, methods, and/or kits for generating and/or using a cell, a cell population, and/or a plurality of cell populations that comprise a polynucleotide encoding and/or a polypeptide comprising a CAR or portion thereof.
  • Any suitable CAR can be used.
  • the CAR comprises a CAR as described in the CARs section below.
  • Any suitable cell can be used.
  • the cell comprises a human cell, such as a human immune cell, for example a neutrophil, eosinophil, basophil, mast cell, monocyte, macrophage, dendritic cell, natural killer cell, a lymphocyte, or a combination thereof, preferably a T cell, and/or a human stem cell, for example a human pluripotent, multipotent stem cell, embryonic stem cell, induced pluripotent stem cell, hematopoietic stem cell, a CD34+ cell, or a combination thereof, preferably a hematopoietic stem cell, more preferably a CD34+ stem cell, even more preferably an induced pluripotent stem cell (iPSC).
  • a human immune cell for example a neutrophil, eosinophil, basophil, mast cell, monocyte, macrophage, dendritic cell, natural killer cell, a lymphocyte, or a combination thereof, preferably a T cell
  • a human stem cell for example a human pluripotent,
  • the cell comprises an allogeneic cell. Any suitable allogeneic cell can be used.
  • the term “allogeneic” includes cell from the same species that are genetically dissimilar and hence immunologically incompatible with the host.
  • the allogeneic cell comprises one or more genetic modifications that reduces the immunogenicity of the cell in the host, e.g., recipient.
  • a cell can be engineered to comprise one or more genomic modifications.
  • the cell can be engineered to comprise one or more genomic modifications that reduce the immunogenicity of the cells, e.g., the modified cell results in little to no immune response in vitro and/or in vivo.
  • an allogeneic cell with respect to a host can be engineered to comprise one or more genomic modifications that reduce the immunogenicity of the one or more allogeneic cells in the host.
  • the cell can be engineered to elicit no more than 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of the immune response as compared to an un-engineered equivalent.
  • the cell can be engineered to elicit no immune response in a host.
  • the immune response can be measured using any suitable technique, for example, flow cytometry or an ELISA.
  • the cell comprises one or more genomic modifications that reduce the immunogenicity of the cell. Any suitable genomic modification and/or combination of genomic modifications thar reduce the immunogenicity of the cell can be used.
  • the cell comprises (1) one or more genomic modifications that partially or completely inactivates one or more genes that codes for a subunit of an HLA-1 protein, (2) one or more genomic modifications that partially or completely inactivates one or more genes coding for a subunit of an HLA-2 protein or a transcription factor regulating the expression of one or more subunits of an HLA-2 protein, and/or (3) one or more genomic modifications that partially or completely inactivates one or more genes that codes for a subunit of a TCR protein.
  • the cell comprises all three genomic modifications.
  • the one or more genomic modifications completely inactivates the one or more genes.
  • the one or more genomic modifications at least partially or completely eliminates surface expression of active (immunogenic) proteins.
  • the one or more genomic modifications completely eliminates surface expression of active (immunogenic) proteins.
  • the cell comprising the one or more genomic modifications can further comprise one or more additional modifications including, but not limited to, introduction of one or more heterologous genes, e.g., transgenes.
  • the one or more transgenes can be introduced into any suitable location in the genome.
  • the one or more transgenes are introduced into a safe harbor site (SHS), e.g., a safe harbor, as discussed in the Genomic safe harbors section below.
  • the one or more transgenes are introduced into one or more of the sites comprising a genomic modification (1) through (3), for example, a CAR transgene can be introduced into one or more genes coding for a subunit of a TCR protein, e.g., a TRAC gene, and/or a B2M-HLA-E and/or a B2M HLA-G fusion protein can be introduced into one or more genes coding for a subunit of an HLA-1 protein, e.g., a B2M gene.
  • Cells can be engineered using any suitable composition and method.
  • a cell can be engineered by delivering to the cell a composition comprising a site- specific nuclease and/or one or more polynucleotides encoding the site-specific nuclease.
  • the site-specific nuclease can be any suitable nuclease, such as a homing endonuclease, a TALEN, a meganuclease, an argonaut, and/or a CRISPR/Cas nuclease, i.e., a nucleic acid-guided nuclease.
  • the site-specific nuclease comprises a nucleic acid-guided nuclease.
  • the site-specific nuclease can hydrolyze the backbone, i.e., generate one or more cuts or strand breaks, in a polynucleotide, e.g., genome, at or near the nuclease's recognition site, i.e., the target site.
  • the one or more strand breaks in at least one strand of the polynucleotide can be repaired via any suitable innate cell repair mechanism, such as non-homologous recombination (NHEJ) and/or homology directed repair (HDR).
  • NHEJ non-homologous recombination
  • HDR homology directed repair
  • repair of one or more strand breaks in at least one strand of the polynucleotide by NHEJ results in one or more genomic modifications, such as insertions and/or deletions (INDELS).
  • one or more portions of heterologous DNA e.g., donor template, can be introduced into the cells and at least a portion of the heterologous DNA can be inserted by the cell at or near the one or more strand breaks in the DNA by HDR.
  • the site-specific nuclease comprises a nucleic acid-guided nuclease, e.g., a CRISPR/Cas nuclease.
  • nucleic acid-guided nuclease can be used to generate one or more strand breaks in a target polynucleotide.
  • the nucleic acid-guided nuclease comprises one or more engineered, non-naturally occurring components.
  • nucleic acid-guided nuclease comprises a Class 1 or Class 2 Cas nuclease, such as a Type V-A, V-B, V-C, V-D, or V-E.
  • the nucleic acid-guided nuclease comprises an amino acid sequence at least 80, 85, 90, 95, 99, or 100% identical to an amino acid sequence of a MAD, ART, or ABW nuclease, such as a MAD1, MAD2, MAD3, MAD4, MAD5, MAD6, MAD7, MAD8, MAD9, MAD10, MAD11, MAD12, MAD13, MAD14, MAD15, MAD16, MAD17, MAD18, MAD19, MAD20, ART1, ART2, ART3, ART4, ART5, ART6, ART7, ART8, ART9, ART10, ART11, ART11*, ART12, ART13, ART14, ART15, ART16, ART17, ART18, ART19, ART20, ART21, ART22, ART23, ART24, ART25, ART26, ART27, ART28, ART29, ART
  • the nucleic acid-guided nuclease comprises an amino acid sequence at least 80, 85, 90, 95, 99, or 100% identical, to the amino acid sequence of MAD2, MAD7, ART2, ART11, or ART11*, even more preferably an amino acid sequence that is at least 880, 85, 90, 95, 99, or 100% identical, to the amino acid sequence of SEQ ID NO: 37.
  • the nucleic acid-guided nuclease comprises one or more nuclear localization signals (NLS), for example 1, 4, or 5 nuclear localization signals, such as 1-5 NLS at the carboxy terminus, 1-5 NLS at the amino terminus, or a combination thereof, preferably one N-terminal NLS and three C-terminal NLS, more preferably five N-terminal NLS.
  • NLS nuclear localization signals
  • Any suitable NLS sequence can be used, for example, SEQ ID NOs: 40-56, preferably any one of SEQ ID NOs: 40, 51, and 56. Any suitable combination of NLS sequences may be used. Additional nucleases and modifications thereof may be found in the Cas proteins section below.
  • the nucleic acid-guided nuclease further comprises a guide nucleic acid.
  • the guide nucleic acid comprises a targeter nucleic acid and a modulator nucleic acid, e.g., a dual guide nucleic acid.
  • the targeter nucleic acid comprises a targeter nucleic acid comprising a targeter stem sequence and a spacer sequence.
  • the modulator nucleic acid comprises a modulator stem sequence complementary to the targeter stem sequence, and, optionally, a 5' sequence.
  • the guide nucleic acid comprises a single polynucleotide.
  • the guide nucleic acid comprises an engineered, non-naturally occurring guide nucleic acid.
  • the guide nucleic acid comprises a dual guide nucleic acid (as described in the Guide nucleic acids section below), wherein the targeter nucleic acid and the modulator nucleic acid are separate polynucleotides.
  • the guide nucleic acid is a dual guide nucleic acid, the stem of the targeter nucleic acid and the stem of the modulator nucleic acid hybridize.
  • the dual guide nucleic acid is capable of binding to and activating a nucleic acid-guided nuclease, that, in a naturally occurring system, is activated by a single cRNA in the absence of a tracrRNA.
  • the guide nucleic acid comprises one or more chemical modifications to one or more nucleotides and/or internucleotide linkages at or near the 5' end, the 3' end, and/or both as described in the gNA modifications section below, such as a 2'-O-alkyl, a 2'-O-methyl, a phosphorothioate, a phosphonoacetate, a thiophosphonoacetate, a 2'-O-methyl-3'- phosphorothioate, a 2'-O-methyl-3'-phosphonoacetate, a 2'-O-methyl-3'-thiophosphonoacetate, a 2'-deoxy-3'-phosphonoacetate, a 2'-deoxy-3'-thiophosphonoacetate, or a combination thereof.
  • a 2'-O-alkyl such as a 2'-O-alkyl, a 2'-O-methyl, a phosphorothioate, a
  • the one or more guide nucleic acids can be complexed with one or more nucleases, e.g., a nucleic acid-guided nuclease complex.
  • the one or more guide nucleic acids, one or more nucleic acid guided nucleases, and/or the one or more nucleic acid-guided nucleases may further comprise a one or more additives that stabilize the nucleic acid-guided nuclease complex.
  • Such cells and/or populations of cells comprising CARs can be used for a variety of purposes, one such purpose can be a CAR T cell. A.
  • compositions, methods, and/or kits comprising CARs or portions thereof ( Figure 3 (302) and (303)).
  • the compositions, methods, and/or kits comprise a dual CARs, e.g., a CAR fusion protein or two separate CARs ( Figure 3 (304) and Figure 4).
  • dual CAR includes a polypeptide comprising a first CAR or portion thereof and a second CAR or portion thereof, either separate, or connected via one or more polypeptide linkers.
  • the second CAR or portion thereof targets the same antigen as the first CAR or portion thereof.
  • the second CAR or portion thereof targets a different antigen than the first CAR or portion thereof.
  • polypeptides comprising any number of CARs or portions thereof, separate or connected via one or more polypeptide linkers.
  • a cell can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 and/or no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 CARs or portions thereof, for example 1-15, preferably 1-10, more preferably, 2-10, even more preferably 2-7, yet more preferably 2-5 CARs or portions thereof, separately or connected via one or more polypeptide linkers.
  • the polypeptide linker can comprise any suitable linker comprising natural or unnaturally occurring amino acids.
  • the CAR or portion thereof is expressed on the cell surface. Additionally or alternatively, the CAR or portion thereof is secreted. [0025] In certain embodiments, the CAR or portion thereof comprises a polypeptide that binds to a binding partner comprising B7H3, BCMA, GPRC5D, CD8, CD8a, CD19, CD20, CD22, CD28, 4-1BB, CD3zeta, a portion thereof, or a combination thereof, preferably B7H3, BCMxA, GPRC5D, CD8, CD8a, CD20, CD22, CD28, 4-1BB, or CD3zeta.
  • a binding partner comprising B7H3, BCMA, GPRC5D, CD8, CD8a, CD19, CD20, CD22, CD28, 4-1BB, CD3zeta, a portion thereof, or a combination thereof, preferably B7H3, BCMxA, GPRC5D, CD8, CD8a, CD20, CD22, CD28, 4-1BB, or CD
  • the CAR or portion thereof is at least 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 86-124 or 2044- 2070 of Table 1, preferably a polypeptide at least 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 86-104, 116-124, or 2044-2070 of Table 1, yet more preferably at least 95% identical, still more preferably least 99% identical.
  • compositions, methods, and/or kits comprising a polynucleotide encoding a polypeptide comprising a CAR or portion thereof.
  • the polynucleotide can be inserted into any suitable location in the genome, for example a safe harbor site (as discussed in the Genomic safe harbors section below) and/or a suitable gene such as a TRAC gene.
  • the polynucleotide can comprise a first portion comprising a first CAR or portion thereof and a second portion comprising a second CAR or portion thereof.
  • the first portion of the polynucleotide and the second portion of the polynucleotide can be separately expressed, for example, a first mRNA transcript generated encoding the first CAR or portion thereof, and a second mRNA transcript generated encoding the second CAR or portion thereof.
  • the first portion and second portion can be expressed as a single mRNA transcript, for example comprising an internal ribosome entry site.
  • Any suitable genomic construct for expression can be used in combination with any suitable number of CARs and/or dual CARs.
  • the CAR or dual CAR or portion thereof can comprise any suitable form, such as an antibody, a nanobody (VHH), a Fab, a scFv, diabody, a triabody, a minibody, a single-domain antibody, a first-generation CAR, a second-generation CAR, a third-generation CAR, and/or a fourth-generation CAR.
  • Any suitable antigen binding domain can be combined with any suitable combination of additional CAR domains as necessary for the application.
  • the CAR or portion thereof can be an antibody, an antigen- binding fragment of an antibody, or a fusion protein derived from such an antibody, such as a single-chain variable fragment (scFv).
  • a scFv can include a single chain Fv antibody in which the variable domains of the heavy chain and of the light chain of a traditional two chain antibody have been joined to form a single polypeptide chain.
  • single chain antibodies contemplated herein can be derived from any species including human or animal (e.g., mice, rabbit, pig, dog, cow, horse, goat, camel, or other animal).
  • the intracellular signaling domain can include a signaling domain and a co-stimulatory domain.
  • the CAR or portion thereof further comprises a spacer domain which links an antigen binding domain to a transmembrane domain.
  • a spacer domain of appropriate length can improve mobility of an antigen binding domain to allow for optimal binding to a target antigen and improve flexibility.
  • the spacer domain comprises at least a portion or segment of a hinge region of an IgGl, IgG2, IgG3, or IgG4.
  • a spacer domain can be derived from a CH2 region and/or CH3 region of an IgGl, IgG2, IgG3, or IgG4.
  • the spacer domain comprises upper hinge amino acids found between the variable heavy chain and the core, and the core hinge amino acids including a polyproline region.
  • the spacer region comprises at least a portion of a hinge region of a human IgG4 hinge spacer. In some embodiments, the spacer region comprises a human IgG4 hinge-CH3 spacer. [0030] In some embodiments, the CAR or portion thereof further comprises a transmembrane domain. A transmembrane domain can provide anchoring of a CAR in a cell membrane. In some embodiments, the transmembrane domain comprises a membrane-bound or transmembrane protein.
  • the transmembrane domain comprises a transmembrane region of an alpha, beta, or zeta chain of a T-cell receptor, such as CD28, CD3, CD45, CD4, CD8, CD8a CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154.
  • the transmembrane domain comprises a CD28 transmembrane domain (CD28tm).
  • the CAR or portion thereof further comprises an intracellular signaling domain linked to a transmembrane domain.
  • the intracellular signaling domain can activate a function of a cell when the antigen binding domain binds to a target antigen.
  • the intracellular signaling domain can activate a function of a cell expressing a CAR, such as a T cell expressing the CAR.
  • the intracellular signaling domain comprises one or more intracellular signaling domains.
  • the intracellular signaling domain comprises a functional domain of a primary cytoplasmic signaling protein.
  • the intracellular signaling domain comprises a functional domain of a primary cytoplasmic signaling protein, and at least one functional domain of one or more secondary cytoplasmic signaling proteins.
  • a primary cytoplasmic signaling protein that acts in a stimulatory manner can contain signaling motifs which are known as intracellular receptor tyrosine-based activation motifs (ITAMs).
  • ITAMs intracellular receptor tyrosine-based activation motifs
  • examples of ITAMs containing primary cytoplasmic signaling domains for use herein include, but are not limited to, those derived from CD3zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d.
  • the intracellular signaling domain and/or the co-stimulatory domain herein can include all or a biologically active fragment of CD27, CD28, 4-1BB, 0X40, CD30, CD40, ICOS, lymphocyte function-associated antigen- 1 (LFA-l), CD2, CD7, LIGHT, NKG2C, or B7H3, and/or a ligand that specifically binds with CD83.
  • the intracellular signaling domain herein can include all or a biologically relevant segment of the signaling domain of CD3-zeta or variant thereof and all or a portion of the signaling domain of 4- 1BB or variant thereof. TABLE 1: CARs B.
  • compositions comprising a polypeptide.
  • the polypeptide comprises a CAR or portion thereof.
  • the polypeptide comprises a sequence that is at least 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, or 100% identical to any one of SEQ ID NOs: 86-124 or 2044-2070, preferably at least 90% identical, more preferably at least 95% identical, even more preferably at least 99% identical, still more preferably 99.5% identical, yet more preferably 100% identical.
  • the polypeptide comprises a sequence that is at least 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, or 100% identical to any one of SEQ ID NOs: 86-104, 116-124, or 2044-2070, preferably at least 90% identical, more preferably at least 95% identical, even more preferably at least 99% identical, still more preferably 99.5% identical, yet more preferably 100% identical.
  • compositions comprising a polynucleotide encoding a polypeptide.
  • the polynucleotide encodes a polypeptide comprising a CAR or portion thereof.
  • the polynucleotide encodes a polypeptide comprising a sequence that is at least 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, or 100% identical to any one of SEQ ID NOs: 86-124 or 2044-2070, preferably at least 90% identical, more preferably at least 95% identical, even more preferably at least 99% identical, still more preferably 99.5% identical, yet more preferably 100% identical.
  • the polynucleotide encodes a polypeptide comprising a sequence that is at least 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, or 100% identical to any one of SEQ ID NOs: 86-104, 116-124, or 2044-2070, preferably at least 90% identical, more preferably at least 95% identical, even more preferably at least 99% identical, still more preferably 99.5% identical, yet more preferably 100% identical.
  • the polynucleotide encodes two or more polypeptides.
  • the polynucleotide comprises a first portion and a second portion, wherein the first portion encodes for a first polypeptide comprising a first CAR or portion thereof and the second portion encodes for a second polypeptide comprising a second CAR or portion thereof.
  • the polynucleotide can encode any suitable number of polypeptides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 and/or no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 polypeptides, for example 1-15, preferably 1-10, more preferably, 2-10, even more preferably 2- 7, yet more preferably 2-5.
  • the polypeptides are separate.
  • the polypeptides are linked.
  • any suitable linker can be used, for example a linker comprises one or more amino acids or a polypeptide, for example a self-cleaving polypeptide.
  • the polypeptide linker can comprise any suitable linker comprising natural or unnaturally occurring amino acids.
  • the first CAR or portion thereof binds to a first site on a first binding partner, e.g., antigen or epitope, and the second CAR or portion thereof binds to a second site on a second binding partner.
  • the first and second sites are the same site on the same binding partner.
  • the first and second sites are different sites on the same binding partner.
  • the first site binds to a site on a first binding partner and the second site binds to a site on a second binding partner.
  • compositions comprising a cell comprising a first polynucleotide encoding a first polypeptide.
  • the first polynucleotide can be inserted into any suitable location in the genome, such as a safe harbor site or a gene, for example a TRAC gene.
  • the first polynucleotide encoding a first polypeptide comprises a first CAR or portion thereof.
  • the first CAR or portion thereof can comprise any suitable CAR or portion thereof as described in the CARs section above.
  • the first polypeptide comprising a CAR or portion thereof can be expressed on the surface of the cell or secreted. In preferred embodiments, the first polypeptide comprising a CAR or portion thereof is expressed on the surface of the cell.
  • An illustrative example is shown in Figure 3. Specifically, Figure 3 shows a first cell comprising a first polypeptide comprising a first CAR or portion thereof (302) and a second cell comprising a second polypeptide comprising a second CAR or portion thereof (303), wherein the second CAR or portion thereof is different from the first CAR or portion thereof.
  • the cell can comprise any suitable number of polypeptides comprising CARs or portions thereof, such as at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 and/or no more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 CARs, for example 1-10 polypeptides or portions thereof, preferably 1-5 polypeptides or portions thereof, more preferably 1-5 polypeptides or portions thereof, even more preferably 1-3 polypeptides or portions thereof. It is to be understood that the cells as depicted comprise polynucleotides encoding the first and/or second polypeptides or portions thereof.
  • the first polynucleotide further encodes for a second polypeptide comprising a second CAR or portion thereof, e.g., a dual CAR.
  • the first CAR or portion thereof can comprise any suitable CAR or portion thereof as described in the CARs section above.
  • the first and/or second polypeptides can be expressed on the surface of the cell or secreted.
  • the first and second polypeptides comprise the same CAR or portion thereof.
  • the second CAR or portion thereof is different from the first CAR or portion thereof.
  • the cell comprising a first polynucleotide further comprises a second polynucleotide of portion thereof encoding a second polypeptide comprising a second CAR or portion thereof, e.g., a dual CAR.
  • the second CAR or portion thereof can comprise any suitable CAR or portion thereof as described in the CARs section above.
  • the second CAR or portion thereof can be expressed on the surface of the cell or secreted.
  • the first and second polypeptides comprise the same CAR or portion thereof.
  • the second CAR or portion thereof is different from the first CAR or portion thereof.
  • the polypeptide comprising a CAR or portion thereof comprises a dual CAR comprising a first CAR or portion thereof linked to a second CAR or portion thereof via a polypeptide linker.
  • the first and second CAR or portions thereof can comprise any suitable CAR or portion thereof as described in the CARs section above.
  • the dual CAR can be expressed on the surface of the cell or secreted.
  • the first and second CARs or portions thereof comprise the same CAR or portion thereof.
  • the second CAR or portion thereof is different from the first CAR or portion thereof.
  • Figure 4 shows a cell comprising an intracellular (401) and extracellular (402) space separated by a cell surface, e.g., cell membrane, comprising a first polypeptide comprising a first dual CAR (403 and 404) and a second polypeptide comprising a second dual CAR (405 and 406) expressed on the surface of the cell, wherein the first dual CAR comprises a second CAR or portion thereof (404) linked to a first CAR or portion thereof (403) and the second dual CAR comprises a first CAR or portion thereof (403) linked to a second CAR or portion thereof (404).
  • a cell surface e.g., cell membrane
  • the first dual CAR comprises a second CAR or portion thereof (404) linked to a first CAR or portion thereof (403)
  • the second dual CAR comprises a first CAR or portion thereof (403) linked to a second CAR or portion thereof (404).
  • the cell can comprise any suitable number of polypeptides comprising dual CARs, such as at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 and/or no more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 polypeptides, for example 1-10 polypeptides, preferably 1-5 polypeptides, more preferably 1-5 polypeptides, even more preferably 1-3 polypeptides.
  • the dual CAR can comprise any suitable combination of CARs or portions thereof. Additional dual CARs, e.g., the second, third, etc. dual CARs, can comprise any suitable combination of CARs or portions thereof different from the first and/or additional dual CARs.
  • the first and/or second polypeptides comprising a first and/or second CAR or portion thereof or dual CAR can be secreted.
  • An illustrative example is shown in Figure 5.
  • Figure 5 shows a cell comprising an intracellular space (501) and an extra cellular space (502) separated by a cell surface, e.g., cell membrane, comprising a first polypeptide comprising a first CAR or portion there (503) expressed on the surface of the cell and a second polypeptide comprising a second CAR or portion thereof (504) expressed in the intracellular space of the cell (501) and secreted to the extracellular space of the cell (502).
  • a cell surface e.g., cell membrane
  • the cell can comprise any suitable number of polypeptides comprising a CAR or portion thereof that is secreted, such as at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 and/or no more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 secreted polypeptides, for example 1-10 secreted polypeptides, preferably 1-5 secreted polypeptides, more preferably 1-5 secreted polypeptides, even more preferably 1-3 secreted polypeptides.
  • the cell comprises a human cell, such as a human immune cell, for example a neutrophil, eosinophil, basophil, mast cell, monocyte, macrophage, dendritic cell, natural killer cell, a lymphocyte, or a combination thereof, preferably a T cell, and/or a human stem cell, for example a human pluripotent, multipotent stem cell, embryonic stem cell, induced pluripotent stem cell, hematopoietic stem cell, a CD34+ cell, or a combination thereof, preferably a hematopoietic stem cell, more preferably a CD34+ stem cell, even more preferably an induced pluripotent stem cell (iPSC).
  • a human immune cell for example a neutrophil, eosinophil, basophil, mast cell, monocyte, macrophage, dendritic cell, natural killer cell, a lymphocyte, or a combination thereof, preferably a T cell
  • a human stem cell for example a human pluripotent,
  • the cell comprises an allogeneic cell.
  • D Cell populations comprising CARs
  • a cell comprises two or more polynucleotides
  • compositions comprising one or more cell populations comprising a first polynucleotide encoding a first polypeptide comprising a first CAR or portion thereof and/or dual CAR and/or a second polynucleotide encoding a second polypeptide comprising a second CAR or portion thereof and/or dual CAR.
  • the composition comprises a single cell population, wherein each of the cells both the first and second polynucleotides.
  • compositions comprising a plurality of cell populations, wherein each cell population comprises a different set of polynucleotides.
  • at least one cell population comprises both the first and second polynucleotides, in addition to one or more additional cell populations that do not comprise both the first and second polynucleotides.
  • the cell population can comprise any one of the cells as described in the Cells comprising polypeptides comprising CARs or portions thereof section above. An illustrative example is shown in Figure 3.
  • Figure 3 shows a target cell (301) that does not comprise either the first or second polynucleotide, whereby introduction into a suitable location in the genome of the first and second polynucleotides using a suitable genome engineering technique results in four possible populations of cells: (301) a cell that where neither the first or second polynucleotide was introduced into suitable locations in the genome; (302) a cell where the first but not the second polynucleotide was introduced intro a suitable location in the genome; (303) a cell where the second but not the first polynucleotide was introduced into a suitable location in the genome; and (304) a cell where both the first and the second polynucleotides were introduced into suitable locations in the genome.
  • the plurality of cell populations comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, or 45 and/or no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 populations, for example 1-50 cell populations, preferably 1-20 cell populations.
  • each cell population in the plurality of cell populations can be present at any percentage relative to the other cell populations, wherein the relative percentage of each population is affected by a number of factors including but not limited to delivery efficiency of the editing components, quality of the editing components, concentration of the editing components, relative efficiency and specificity of the editing events, vitality of the cells, and/or viability of the cells before or after introduction of the one or more genomic modifications.
  • the first cell population comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70% and/or no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 75% of all of the cells in the plurality of cell populations, for example 1-75% of all the cells in the plurality of cell populations, preferably 5- 75%, more preferably 10-75%, even more preferably 15-75%, yet even more preferably 20-75%.
  • the second cell population comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70% and/or no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 75% of all of the cells in the plurality of cell populations, for example 1-75% of all the cells in the plurality of cell populations, preferably no more than 50%, more preferably no more that 30%, even more preferably no more than 20%, yet even more preferably no more than 10%.
  • the third cell population comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70% and/or no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 75% of all of the cells in the plurality of cell populations, for example 1-75% of all the cells in the plurality of cell populations preferably no more than 50%, more preferably no more that 30%, even more preferably no more than 20%, yet even more preferably no more than 10%.
  • the fourth cell population comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70% and/or no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 75% of all of the cells in the plurality of cell populations, for example 1-75% of all the cells in the plurality of cell populations, preferably no more than 50%, more preferably no more that 30%, even more preferably no more than 20%, yet even more preferably no more than 10%. It is understood that the sum of the percentages for each cell population in the plurality adds to 100%.
  • the number, relative abundance, and/or identity of cell populations in a plurality of cell populations can be measured by any suitable method.
  • the number, relative abundance, and/or identity of cell populations in a plurality of cell populations can be measured by analyzing one or more nucleic acids in a sample using one or more methods, for example PCR, multiplex PCR, FISH, and/or sequencing.
  • the number and/or identity of cell populations in a plurality of cell populations can be measured by analyzing one or more cell surface proteins and/or lack thereof in a sample using one or more methods, for example immunostaining and microscopy, ELISA, pull downs, and/or flow cytometry.
  • compositions and/or kits for engineering cells to comprise CARs comprising a guide nucleic acid, a nucleic acid-guided nuclease, a nucleic acid-guided nuclease complex, and/or one or more polynucleotides encoding thereof.
  • the composition further comprises a donor template.
  • the composition further comprises an additive that stabilized the nucleic acid-guided nuclease complex, such as poly-L-glutamic acid.
  • the one or more components of the composition are combined in the presence of an aqueous buffer.
  • the composition further comprises an excipient.
  • the composition is dehydrated, e.g., freeze-dried or lyophilized.
  • compositions comprising a first nucleic acid-guided nuclease, a first guide nucleic acid, and/or a first donor template comprising a first polynucleotide encoding a first CAR or portion thereof.
  • the composition further comprises a second nucleic acid-guided nuclease, a second guide nucleic acid, and/or a second donor template comprising a second polynucleotide encoding a second CAR or portion thereof.
  • the composition further comprises a third nucleic acid-guided nuclease, a third guide nucleic acid, and/or a third donor template comprising a third polynucleotide encoding a third CAR or portion thereof.
  • a third nucleic acid-guided nuclease N
  • gNA guide nucleic acid
  • D donor template
  • the composition comprises a first nucleic acid-guided nuclease (N) 1 , a first guide nucleic acid (gNA) 1 , and/or a first donor template (D) 1 .
  • x can be any suitable integer, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40 and/or no more than 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2, wherein the composition the suitable number of each component.
  • the plurality of genomic modifications, 2 or more can be generated sequentially. In certain embodiments, the plurality of genomic modifications can be generated simultaneously. Methodology is further discussed in the Methods for engineering cells to comprise CARs section below.
  • the nucleic acid-guided nuclease can be any suitable nuclease, such as a homing endonuclease, a TALEN, a meganuclease, an argonaut, and/or a CRISPR/Cas nuclease, preferably a CRISPR/Cas nuclease, more preferably a Class 1 or Class 2 Cas nuclease, even more preferably a Class 2 nuclease, yet more preferably a Type V-A, V-B, V-C, V-D, or V-E, still more preferably a Type V-A nuclease.
  • a CRISPR/Cas nuclease preferably a Class 1 or Class 2 Cas nuclease, even more preferably a Class 2 nuclease, yet more preferably a Type V-A, V-B, V-C, V-D, or V-E, still more preferably a Type V-
  • the nucleic acid-guided nuclease comprises an amino acid sequence at least 80, 85, 90, 95, 99, or 100% identical to an amino acid sequence of a MAD, ART, or ABW nuclease, such as a MAD1, MAD2, MAD3, MAD4, MAD5, MAD6, MAD7, MAD8, MAD9, MAD10, MAD11, MAD12, MAD13, MAD14, MAD15, MAD16, MAD17, MAD18, MAD19, MAD20, ART1, ART2, ART3, ART4, ART5, ART6, ART7, ART8, ART9, ART10, ART11, ART11*, ART12, ART13, ART14, ART15, ART16, ART17, ART18, ART19, ART20, ART21, ART22, ART23, ART24, ART25, ART26, ART27, ART28, ART29, ART
  • the nucleic acid-guided nuclease comprises one or more nuclear localization signals (NLS), for example 1, 4, or 5 nuclear localization signals, such as 1-5 NLS at the carboxy terminus, 1-5 NLS at the amino terminus, or a combination thereof, preferably one N-terminal NLS and three C-terminal NLS.
  • NLS nuclear localization signals
  • a NLS comprises any one of SEQ ID NOs: 40-56, preferably SEQ ID NOs: 40, 51, and 56. Additional nucleases and modifications thereof may be found in the Cas proteins section below.
  • the guide nucleic acid can be any gNA compatible with the nucleic acid-guided nuclease.
  • the guide nucleic acid comprises a targeter nucleic acid and a modulator nucleic acid, e.g., a dual guide nucleic acid.
  • the targeter nucleic acid comprises a targeter nucleic acid comprising a targeter stem sequence and a spacer sequence.
  • the modulator nucleic acid comprises a modulator stem sequence complementary to the targeter stem sequence, and, optionally, a 5' sequence.
  • the guide nucleic acid comprises a single polynucleotide.
  • the guide nucleic acid comprises an engineered, non-naturally occurring guide nucleic acid.
  • the guide nucleic acid comprises a dual guide nucleic acid (as described in the Guide nucleic acids section below), wherein the targeter nucleic acid and the modulator nucleic acid are separate polynucleotides.
  • the guide nucleic acid is a dual guide nucleic acid
  • the stem of the targeter nucleic acid and the stem of the modulator nucleic acid hybridize.
  • the dual guide nucleic acid is capable of binding to and activating a nucleic acid-guided nuclease, that, in a naturally occurring system, is activated by a single cRNA in the absence of a tracrRNA.
  • the guide nucleic acid comprises a donor recruiting sequence.
  • the gNA can comprise any suitable spacer sequence.
  • the gNA comprises a spacer sequence as shown in Table 2.
  • the guide nucleic acid comprises one or more chemical modifications to one or more nucleotides and/or internucleotide linkages at or near the 5' end, the 3' end, and/or both as described in the gNA modifications section below, such as a 2'-O-alkyl, a 2'-O-methyl, a phosphorothioate, a phosphonoacetate, a thiophosphonoacetate, a 2'-O-methyl-3'- phosphorothioate, a 2'-O-methyl-3'-phosphonoacetate, a 2'-O-methyl-3'-thiophosphonoacetate, a 2'-deoxy-3'-phosphonoacetate, a 2'-deoxy-3'-thiophosphonoacetate, or a combination thereof
  • the one or more guide nucleic acids can be complexed with one or more nucleases, e.g., a nucleic acid-guided nuclease complex.
  • the one or more guide nucleic acids, one or more nucleic acid guided nucleases, and/or the one or more nucleic acid-guided nucleases may further comprise a one or more additives that stabilize the nucleic acid-guided nuclease complex.
  • An illustrative example of a nucleic acid-guided nuclease complex is shown in Figure 6.
  • Figure 6 shows a Type V-A nucleic acid guided nuclease (601) complexed with a dual gNA comprising a modulator nucleic acid (606) and a targeter nucleic acid (607), wherein the modulator nucleic acid and targeter nucleic acid are hybridized through a stem.
  • the targeter nucleic acid further comprises a spacer sequence (605) at least partially complementary to a target nucleotide sequence (604), i.e., a protospacer, in a target polynucleotide (602) adjacent to a suitable PAM (603).
  • the nucleic acid-guided nuclease complex Upon binding to the target nucleotide sequence, the nucleic acid-guided nuclease complex can generate one or more strand breaks (608) in the target polynucleotide at or near the target nucleotide sequence.
  • Any suitable donor template can be used as described in the Donor templates section below.
  • the donor template comprises a polynucleotide encoding a polypeptide, wherein the polypeptide comprises a chimeric antigen receptor (CAR) or portion thereof.
  • the polynucleotide can encode for any suitable number of CARs or dual CARs as described above.
  • the donor template comprises one or more chemical modifications to one or more nucleotides and/or internucleotide linkages at or near the 5' end, the 3' end, and/or both as described in the gNA modifications section below, such as a 2'-O-alkyl, a 2'-O-methyl, a phosphorothioate, a phosphonoacetate, a thiophosphonoacetate, a 2'-O-methyl-3'- phosphorothioate, a 2'-O-methyl-3'-phosphonoacetate, a 2'-O-methyl-3'-thiophosphonoacetate, a 2'-deoxy-3'-phosphonoacetate, a 2'-deoxy-3'-thiophosphonoacetate, or a combination thereof.
  • a 2'-O-alkyl such as a 2'-O-alkyl, a 2'-O-methyl, a phosphorothioate, a phosphono
  • Methods for engineered cells to comprise CARs [0054] In certain embodiments, provided herein are methods. In certain embodiments, provided herein are methods for engineering cells, such as human cells. In certain embodiments, the methods are for engineering cells to comprise one or more CARs or dual CARs. In certain preferred embodiments, provided herein are methods using any composition as described in the Compositions and/or kits for engineering cells to comprise CARs section above to generate any composition as described in the Cells comprising polypeptides comprising CARs or portions thereof or the Cell populations comprising CARs sections above.
  • a nucleic acid-guided nuclease, a guide nucleic acid, a nucleic acid-guided nuclease complex, one or more polynucleotides encoding thereof, a donor template, and/or a suitable combination thereof are delivered into the cell, wherein the one or more components are transferred to the nuclease, whereby one or more genomic modifications is generated.
  • An exemplary method is shown in Figure 7. Specifically Figure 7A shows a nucleic acid-guided nuclease complex (701) and a donor template (702) being delivered to the cell (703), wherein the one or more components are transferred to the nuclease (704).
  • the nucleic-acid guide nuclease comprises one or more nuclease localization signals (as described above and in the Ribonucleoprotein (RNP) section below).
  • the nucleic acid-guided nuclease complex comprising one or more nuclear localizations signals (705) and the donor template (706) are delivered to the cell (708), wherein the donor template (706) and nucleic acid-guided nuclease complex (705) bind together (707) and are transferred into the nuclease (709) aided by the one or more nuclear localization signals.
  • the method can generate at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 and/or no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 100 genomic modifications, for example, 1-100 genomic modifications, preferably 1-20 genomic modifications, either simultaneously or sequentially (see Multiplexing section below).
  • a first genomic modification is introduced into one or more target cells, wherein the target cell comprises a wild- type cell or a cell comprising one or more genomic modifications (see Cells comprising polypeptides comprising CARs or portions thereof or the Cell populations comprising CARs sections above).
  • the target cell comprises one or more of the modified cells as described in the Cells comprising polypeptides comprising CARs or portions thereof or the Cell populations comprising CARs sections above.
  • the method comprises generating one or more genomic modifications in one or more target cells, wherein the one or more genomic modifications are generated simultaneously.
  • the method comprises generating one or more genomic modifications in one or more target cells, wherein one or more of the genomic modifications are generated sequentially.
  • the one or more genomic modifications may be introduced in any suitable quantity, order, and/or combination.
  • the three genomic modifications can be introduced in any one of the following orders: (1) A then B then C; (2) A then C then B; (3) A and B then C; (4) A then B and C; (5) A and C then B; (6) A then C and B; (7) B then A then C; (8) B then C then A; (9) B and A then C; (10) B then A and C; (11) B and C then A; (12) B then C and A; (13) C then A then B; (14) C then B then A; (15) C and A then B; (16) C then A and B; (17) C then B and A; (18) C and B then A; or (19) A and B and C.
  • Figure 8A shows a first set of components for generating one or more genomic modifications (801) being delivered to a starting cell (802), whereby a first engineered cell or plurality of cell populations (803) is generated.
  • the first engineered cell or plurality of cell populations (803) can be the final resulting cell or plurality of cell populations (804), and/or an additional set of components for generating one or more genomic modifications (805) can be delivered to (803) resulting in a second engineered cell or plurality of cell populations (806). This process can be repeated any number of times as necessary.
  • a method for introducing a plurality of exogenous nucleic acids into the genome of a target cell comprising contacting the target cell with a composition comprising a nucleic acid-guided nuclease (N) x , a guide nucleic acid (gNA) x , and/or a donor template (D) x , wherein the one or more components of the composition are capable of a initiating host cell mediated recombination, e.g., HDR or homologous recombination, of at least a portion of (D) x at a target site (TS) x selected from a plurality of target sites of the genome of the target cell, wherein, for each target site, (N) x is capable of cleaving at (TS) x resulting in homologous recombination of at least a portion of (D) x , wherein the integer (x) represents a genomic modification to be introduced into a suitable location of the genome
  • the composition comprises a first nucleic acid-guided nuclease (N) 1 , a first guide nucleic acid (gNA) 1 , and/or a first donor template (D) 1 .
  • x can be any suitable integer, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40 and/or no more than 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2, wherein the composition the suitable number of each component.
  • the plurality of genomic modifications, 2 or more can be generated sequentially. In certain embodiments, the plurality of genomic modifications can be generated simultaneously. [0057]
  • provided herein are methods for engineering one or more suitable human cells.
  • the cells comprise one or more suitable human stem cells or human immune cells.
  • the cells comprise one or more human cells comprising an immune cell comprising a neutrophil, eosinophil, basophil, mast cell, monocyte, macrophage, dendritic cell, natural killer cell, a lymphocyte, or a combination thereof.
  • the cells comprise one or more T cells.
  • the cells comprises one or more chimeric antigen receptor (CAR)-T cells.
  • the CAR T cell comprises a CAR or portion thereof.
  • the CAR T cell comprises two or more CAR polypeptides or portions thereof.
  • the CAR T cell comprises two CAR polypeptides or portions thereof, wherein the second CAR polypeptide is different than the first CAR polypeptide.
  • the CAR T cell comprises a dual CAR or portion thereof.
  • the cells comprise one or more human stem cells comprising a human pluripotent, multipotent stem cell, embryonic stem cell, induced pluripotent stem cell, hematopoietic stem cell, a CD34+ cell, a combination thereof.
  • the cells comprise one or more hematopoietic stem cells.
  • the cells comprise one or more CD34+ stem cells.
  • the cells comprise one or more induced pluripotent stem cells (iPSC).
  • the cells comprise an allogeneic cell.
  • the one or more cells comprising one or more introduced genomic modifications are either grown, e.g., expanded, or differentiated, for example an iPSC differentiated into a T cell.
  • the one or more target cells are expanded after introduction of the first set of genomic modifications, wherein the second set of genomic modifications are introduced into the progeny of the first set of cells.
  • the stem cells are differentiated before or after introduction of one or more genomic modifications.
  • the stem cells are differentiated after introduction of one or more genomic modifications. An exemplary method is shown in Figure 8B.
  • the cells may be grown or propagated (807) after introduction of the first set of genomic modifications, wherein the second set of genomic modifications are introduced into the progeny of the cells or plurality of cell populations.
  • the cells may be differentiated (808) at any stage of the engineering process, for example, an iPSC differentiated to an immune cell after receiving a plurality of genomic modifications.
  • one or more genomic modifications are introduced into a population of cells, wherein the resulting cell population comprises a plurality of cell populations each having received a different set of genomic modifications (see Cell populations comprising CARs section above).
  • each cell population in the plurality of cell populations can be present at any percentage relative to the other cell populations, wherein the relative percentage of each population is affected by a number of factors including but not limited to delivery efficiency of the editing components, quality of the editing components, concentration of the editing components, relative efficiency and specificity of the editing events, vitality of the cells, and/or viability of the cells before or after introduction of the one or more genomic modifications.
  • kits for engineering cells comprising delivering one or more site-specific nucleases to the one or more target cells.
  • the one or more site-specific nucleases are delivered to the target cells as a polypeptide.
  • the one or more site-specific nucleases comprise a nucleic acid-guided nuclease system, e.g., a CRISPR/cas system.
  • one or more polynucleotides encoding one or more components of the nuclease system are delivered to the target cells.
  • the nucleic acid-guided nuclease system comprises a Type V nuclease, more preferably a Type V-A nuclease, even more preferably a MAD2, MAD7, ART2, ART11, ART11* nucleases, yet more preferably a MAD7 nuclease.
  • one more guide nucleic acids comprising a spacer sequence at least partially complementary a target nucleotide sequence within a site wherein one or more genomic modifications are to be introduced are delivered to the target cells.
  • one or more nucleic acid-guided nucleases are delivered to the target cells.
  • a combination of one or more guide nucleic acids and nucleic acid-guided nucleases are delivered to the target cells, wherein the one or more nucleic acid-guided nucleases are optionally complexed with a guide nucleic acid (see Ribonucleoprotein (RNP) section below).
  • RNP Ribonucleoprotein
  • one or more fully formed nucleic acid-guided nuclease complexes are delivered, e.g., RNP.
  • any one of the embodiments as described in the Compositions and/or kits for engineering cells to comprise CARs section can be delivered to the target cell. II.
  • a CRISPR-Cas system generally comprises a Cas protein and one or more guide nucleic acids (gNAs).
  • the Cas protein can be directed to a specific location in a double-stranded DNA target by recognizing a protospacer adjacent motif (PAM) in the non-target strand of the DNA, and the one or more guide nucleic acids can be directed to a specific location by hybridizing with a target nucleotide sequence, also referred to herein as a target sequence, in the target strand of the target polynucleotide.
  • PAM protospacer adjacent motif
  • a guide nucleic acid can be designed to comprise a nucleotide sequence called a spacer sequence that is at least partially complementary to and can hybridize with a target nucleotide sequence, where target nucleotide sequence is located adjacent to a PAM in an orientation operable with the Cas protein. It has been observed that not all CRISPR-Cas systems designed by these criteria are equally effective.
  • the larger polynucleotide in which a target nucleotide sequence is located may be referred to as a target polynucleotide; e.g., a chromosome or other genomic DNA, or portion thereof, or any other suitable polynucleotide within which a target nucleotide sequence is located.
  • the target polynucleotide in double stranded DNA comprises two strands.
  • the strand of the DNA duplex to which the spacer sequence is complementary herein is called the “target strand,” while the strand to which the spacer sequence shares sequence identity herein is called the “non-target strand.”
  • Class 1 CRISPR- Cas systems utilize multi-protein effector complexes
  • class 2 CRISPR-Cas systems utilize single-protein effectors
  • type II and type V systems typically target DNA and type VI systems typically target RNA (id.).
  • Naturally occurring type II effector complexes include Cas9, CRISPR RNA (crRNA), and trans-activating CRISPR RNA (tracrRNA), but the crRNA and tracrRNA can be fused as a single guide RNA in an engineered system for simplicity (see, Wang et al. (2016) ANNU. REV. BIOCHEM., 85: 227).
  • Naturally occurring type V systems such as type V-A, type V-C, and type V-D systems, do not require tracrRNA and use crRNA alone as the guide for cleavage of target DNA (see, Zetsche et al. (2015) CELL, 163: 759; Makarova et al. (2017) CELL, 168: 328.
  • Naturally occurring type II CRISPR-Cas systems e.g., CRISPR-Cas9 systems
  • CRISPR-Cas9 systems generally comprise two guide nucleic acids, called crRNA and tracrRNA, which form a complex by nucleotide hybridization.
  • Single guide nucleic acids capable of activating type II Cas nucleases have been developed, for example, by linking the crRNA and the tracrRNA (see, e.g., U.S. Patent Nos.10,266,850 and 8,906,616).
  • Naturally occurring type II Cas proteins comprise a RuvC-like nuclease domain and an HNH endonuclease domain, and recognize a 3’ G-rich PAM located immediately downstream from the target nucleotide sequence, the orientation determined using the non-target strand (i.e., the strand not hybridized with the spacer sequence) as the coordinate.
  • the CRISPR-Cas systems cleave a double-stranded DNA to generate a blunt end.
  • the cleavage site is generally 3-4 nucleotides upstream from the PAM on the non-target strand.
  • Naturally occurring Type V-A, Type V-C, and Type V-D CRISPR-Cas systems lack a tracrRNA and rely on a single crRNA to guide the CRISPR-Cas complex to the target polynucleotide.
  • Dual guide nucleic acids capable of activating type V-A, type V-C, or type V-D Cas nucleases have been developed, for example, by splitting the single crRNA into a targeter nucleic acid and a modulator nucleic acid (see, e.g., International (PCT) Application Publication No. WO 2021/067788).
  • Naturally occurring type V-A Cas proteins comprise a RuvC-like nuclease domain but lack an HNH endonuclease domain, and recognize a 5’ T-rich PAM located immediately upstream from the target nucleotide sequence, the orientation determined using the non-target strand (i.e., the strand not hybridized with the spacer sequence) as the coordinate.
  • These CRISPR-Cas systems cleave a double-stranded DNA to generate a staggered double- stranded break rather than a blunt end.
  • the cleavage site is distant from the PAM site (e.g., separated by at least 10, 11, 12, 13, 14, or 15 nucleotides downstream from the PAM on the non- target strand and/or separated by at least 15, 16, 17, 18, or 19 nucleotides upstream from the sequence complementary to PAM on the target strand).
  • Elements in an exemplary single guide CRISPR Cas system e.g., a type V-A CRISPR-Cas system, are shown in Figure 1A.
  • the single gNA can also be called a “crRNA” or “single gRNA” where it is present in the form of an RNA.
  • an optional 5’ sequence e.g., a tail, a modulator stem sequence, a loop, a targeter stem sequence complementary to the modulator stem sequence, and a spacer sequence that is at least partially complementary to and can hybridize with a target sequence in the target strand of the target polynucleotide.
  • an optional 5’ sequence e.g., a tail, a modulator stem sequence, a loop, a targeter stem sequence complementary to the modulator stem sequence, and a spacer sequence that is at least partially complementary to and can hybridize with a target sequence in the target strand of the target polynucleotide.
  • the sequence including the 5’ tail and the modulator stem sequence can also be called a “modulator sequence” herein.
  • a fragment of the single guide nucleic acid from the optional 5’ tail to the targeter stem sequence also called a “scaffold sequence” herein, bind the Cas protein.
  • the second guide nucleic acid which can be called “targeter nucleic acid” herein, comprises, from 5’ to 3’, a targeter stem sequence complementary to the modulator stem sequence and a spacer sequence that is at least partially complementary to and can hybridize with the target sequence in the target strand of the target polynucleotide.
  • the duplex between the modulator stem sequence and the targeter stem sequence, plus the optional 5’ tail, constitute a structure that binds the Cas protein.
  • the PAM in the non-target strand of the target DNA binds the Cas protein.
  • the duplex formed between the targeter stem sequence and the modulator stem sequence corresponds to the duplex formed between the crRNA and the tracrRNA.
  • the duplex formed between the targeter stem sequence and the modulator stem sequence corresponds to the stem portion of a stem-loop structure in the scaffold sequence of the crRNA. It is understood that 100% complementarity is not required between the targeter stem sequence and the modulator stem sequence.
  • the guide nucleic acid is capable of activating a Cas nuclease.
  • a gNA capable of activating a particular Cas nuclease is said to be “compatible” with the Cas nuclease; a Cas nuclease capable of being activated by a particular gNA is said to be “compatible” with the gNA.
  • CRISPR-Associated protein can refer to a naturally occurring Cas protein or an engineered Cas protein.
  • Non-limiting examples of Cas protein engineering include but are not limited to mutations and modifications of the Cas protein that alter the activity of the Cas, alter the PAM specificity, broaden the range of recognized PAMs, and/or reduce the ability to modify one or more off-target loci as compared to a corresponding unmodified Cas.
  • the Cas protein is a type V-A, type V-C, or type V-D Cas protein. In certain embodiments, the Cas protein is a type V-A Cas protein. In other embodiments, the Cas protein is a type II Cas protein, e.g., a Cas9 protein.
  • a type V-A Cas nucleases comprises Cpf1. Cpf1 proteins are known in the art and are described, e.g., in U.S. Patent Nos.9,790,490 and 10,113,179. Cpf1 orthologs can be found in various bacterial and archaeal genomes.
  • the Cpf1 protein is derived from Francisella novicida U112 (Fn), Acidaminococcus sp. BV3L6 (As), Lachnospiraceae bacterium ND2006 (Lb), Lachnospiraceae bacterium MA2020 (Lb2), Candidatus Methanoplasma termitum (CMt), Moraxella bovoculi 237 (Mb), Porphyromonas crevioricanis (Pc), Prevotella disiens (Pd), Francisella tularensis 1, Francisella tularensis subsp.
  • a type V-A Cas nuclease comprises AsCpf1 or a variant thereof.
  • a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 3 of International (PCT) Application Publication No. WO 2021/158918.
  • a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 3 of International (PCT) Application Publication No. WO 2021/158918.
  • a type V-A Cas nuclease comprises LbCpf1 or a variant thereof.
  • a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 4 of International (PCT) Application Publication No. WO 2021158918.
  • a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 4 of International (PCT) Application Publication No. WO 2021/158918.
  • a type V-A Cas nuclease comprises FnCpf1 or a variant thereof.
  • a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 5 of International (PCT) Application Publication No. WO 2021158918.
  • a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 5 of International (PCT) Application Publication No. WO 2021/158918.
  • a type V-A Cas nuclease comprises Prevotella bryantii Cpf1 (PbCpf1) or a variant thereof.
  • a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 6 of International (PCT) Application Publication No.
  • a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 6 of International (PCT) Application Publication No. WO 2021/158918.
  • a type V-A Cas nuclease comprises Proteocatella sphenisci Cpf1 (PsCpf1) or a variant thereof.
  • a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7 of International (PCT) Application Publication No. WO 2021158918.
  • a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 7 of International (PCT) Application Publication No. WO 2021/158918.
  • a type V-A Cas nuclease comprises Anaerovibrio sp.
  • a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 8 of International (PCT) Application Publication No. WO 2021158918.
  • a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 8 of International (PCT) Application Publication No. WO 2021/158918.
  • a type V-A Cas nuclease comprises Moraxella caprae Cpf1 (McCpf1) or a variant thereof.
  • a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 9 of International (PCT) Application Publication No. WO 2021/158918.
  • a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 9 of International (PCT) Application Publication No.
  • a type V-A Cas nuclease comprises Lachnospiraceae bacterium COE1 Cpf1 (Lb3Cpf1) or a variant thereof.
  • a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 10 of International (PCT) Application Publication No. WO 2021158918.
  • a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 10 of International (PCT) Application Publication No. WO 2021/158918.
  • a type V-A Cas nuclease comprises Eubacterium coprostanoligenes Cpf1 (EcCpf1) or a variant thereof.
  • a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 11 of International (PCT) Application Publication No.
  • a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 11 of International (PCT) Application Publication No. WO 2021/158918.
  • a type V-A Cas nuclease is not Cpf1. In certain embodiments, a type V-A Cas nuclease is not AsCpf1.
  • a type V-A Cas nuclease comprises MAD1, MAD2, MAD3, MAD4, MAD5, MAD6, MAD7, MAD8, MAD9, MAD10, MAD11, MAD12, MAD13, MAD14, MAD15, MAD16, MAD17, MAD18, MAD19, or MAD20, or variants thereof.
  • MAD1-MAD20 are known in the art and are described in U.S. Patent No.9,982,279.
  • a type V-A Cas nuclease comprises MAD7 or a variant thereof.
  • a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 37. In certain embodiments, a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 37.
  • MAD7 (SEQ ID NO: 37) MNNGTNNFQNFIGISSLQKTLRNALIPTETTQQFIVKNGIIKEDELRGENRQILKDIMDDYYRGF ISETLSSIDDIDWTSLFEKMEIQLKNGDNKDTLIKEQTEYRKAIHKKFANDDRFKNMFSAKLISD ILPEFVIHNNNYSASEKEEKTQVIKLFSRFATSFKDYFKNRANCFSADDISSSSCHRIVNDNAEI FFSNALVYRRIVKSLSNDDINKISGDMKDSLKEMSLEEIYSYEKYGEFITQEGISFYNDICGKVN SFMNLYCQKNKENKNLYKLQKLHKQILCIADTSYEVPYKFESDEEVYQSVNGFLDNISSKHIVER LRKIGDNYNGYNLDKIYIVSKFYESVSQKTYRDWETINTALEIHYNNILPGNGKSKADKVKKAVK N
  • a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 38.
  • MAD2 (SEQ ID NO: 38) MSSLTKFTNKYSKQLTIKNELIPVGKTLENIKENGLIDGDEQLNENYQKAKIIVDDFLRDFINKA LNNTQIGNWRELADALNKEDEDNIEKLQDKIRGIIVSKFETFDLFSSYSIKKDEKIIDDDNDVEE EELDLGKKTSSFKYIFKKNLFKLVLPSYLKTTNQDKLKIISSFDNFSTYFRGFFENRKNIFTKKP ISTSIAYRIVHDNFPKFLDNIRCFNVWQTECPQLIVKADNYLKSKNVIAKDKSLANYFTVGAYDY FLSQNGIDFYNNIIGGLPAFAGHEKIQGLNEFINQECQKDSELKSKLKNRHAFKMAVLFKQILSD REKSFVIDEFESDAQVIDAVKNFYAEQCKDNNVIFNLLNLIKNIAFLSDDELDGIFIEGKYLSSV SQKLYSDWSKLRNDIEDSANSKQ
  • Csm1 proteins are known in the art and are described in U.S. Patent No.9,896,696.
  • Csm1 orthologs can be found in various bacterial and archaeal genomes.
  • a Csm1 protein is derived from Smithella sp. SCADC (Sm), Sulfuricurvum sp. (Ss), or Microgenomates (Roizmanbacteria) bacterium (Mb).
  • a type V-A Cas nuclease comprises SmCsm1 or a variant thereof.
  • a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 12 of International (PCT) Application Publication No. WO 2021/158918.
  • a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 12 of International (PCT) Application Publication No. WO 2021/158918.
  • a type V-A Cas nuclease comprises SsCsm1 or a variant thereof.
  • a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 13 of International (PCT) Application Publication No. WO 2021/158918.
  • a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 13 of International (PCT) Application Publication No. WO 2021/158918.
  • a type V-A Cas nuclease comprises MbCsm1 or a variant thereof.
  • a type V-A Cas protein comprises an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 14 of International (PCT) Application Publication No. WO 2021/158918.
  • a type V-A Cas protein comprises the amino acid sequence set forth in SEQ ID NO: 14 of International (PCT) Application Publication No. WO 2021/158918.
  • the type V-A Cas nuclease comprises an ART nuclease or a variant thereof.
  • the Type V-A nuclease comprises an ART1, ART2, ART3, ART4, ART5, ART6, ART7, ART8, ART9, ART10, ART11, ART12, ART13, ART14, ART15, ART16, ART17, ART18, ART19, ART20, ART21, ART22, ART23, ART24, ART25, ART26, ART27, ART28, ART28, ART30, ART31, ART32, ART33, ART34, ART35, or ART11* (i.e., ART11_L679F, i.e., ART11 wherein leucine (L) at amino acid position 679 is replaced with phenylalanine (F
  • nucleic acid-guided nuclease comprising a nucleic acid-guided nuclease polypeptide having at least 85% identity to an amino acid sequence represented by SEQ ID NOs: 1-36 or a nucleic acid encoding a nucleic acid-guided nuclease polypeptide comprising at least 85% identity with the polynucleotide represented by SEQ ID NOs: 1-36.
  • nucleic acid-guided nuclease comprising a polypeptide having at least 90% identity to the amino acid sequence represented by SEQ ID NOs: 1-36, wherein the polypeptide does not contain a peptide motif of YLFQIYNKDF (SEQ ID NO: 39).
  • nucleic acid-guided nuclease comprising a nucleic acid encoding a polypeptide having at least 90% identity to nucleic acids represented by SEQ ID NOs: 808-845 wherein an encoded polypeptide does not contain a peptide motif of YLFQIYNKDF (SEQ ID NO: 39).
  • nucleic acid-guided nuclease wherein the polypeptide comprises at least 90% identity with the amino acid sequence represented by SEQ ID NOs: 1-9. In certain embodiments, provided is a nucleic acid-guided nuclease, wherein the polypeptide comprises a polypeptide comprising at least 90% identity with the amino acid sequence represented by SEQ ID NO: 2, 11, or 36. TABLE 3: ART nucleases
  • a Cas nuclease comprises ABW1 (SEQ ID NO: 3), ABW2 (SEQ ID NO: 16), ABW3 (SEQ ID NO: 29), ABW4 (SEQ ID NO: 42), ABW5 (SEQ ID NO: 55), ABW6 (SEQ ID NO: 68), ABW7 (SEQ ID NO: 81), ABW8 (SEQ ID NO: 94), or ABW9 (SEQ ID NO: 107) (all SEQ ID NOs for ABW1-9 and variants thereof from International (PCT) Application Publication No.
  • WO 2021/108324 or variants thereof, such as any one of variants 1-10 of ABW1 (SEQ ID NOs: 4-13, respectively), any one of variants 1-10 of ABW2 (SEQ ID NOs: 17-26, respectively), any one of variants 1-10 of ABW3 (SEQ ID NOs: 30-39, respectively), any one of variants 1-10 of ABW4 (SEQ ID NOs: 43-52, respectively), any one of variants 1-10 of ABW5 (SEQ ID NOs: 56-65, respectively), any one of variants 1-10 of ABW6 (SEQ ID NOs: 69-78, respectively), any one of variants 1-10 of ABW7 (SEQ ID NOs: 82-91, respectively), any one of variants 1-10 of ABW8 (SEQ ID NOs: 95-104, respectively), any one of variants 1-10 of ABW9 (SEQ ID NOs: 108-117, respectively).
  • More type V-A Cas nucleases and their corresponding naturally occurring CRISPR- Cas systems can be identified by computational and experimental methods known in the art, e.g., as described in U.S. Patent No.9,790,490 and Shmakov et al. (2015) MOL. CELL, 60: 385.
  • Exemplary computational methods include analysis of putative Cas proteins by homology modeling, structural BLAST, PSI-BLAST, or HHPred, and analysis of putative CRISPR loci by identification of CRISPR arrays.
  • the Cas protein is a Cas nuclease that directs cleavage of one or both strands at the target locus, such as the target strand (i.e., the strand having the target nucleotide sequence that is at least partially complementary to and can hybridize with a single guide nucleic acid or dual guide nucleic acids) and/or the non-target strand.
  • the Cas nuclease directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more nucleotides from the first or last nucleotide of the target nucleotide sequence or its complementary sequence.
  • the cleavage is staggered, i.e. generating sticky ends.
  • the cleavage generates a staggered cut with a 5' overhang.
  • the cleavage generates a staggered cut with a 5' overhang of 1 to 5 nucleotides, e.g., of 4 or 5 nucleotides.
  • a composition provided herein comprises a Cas nuclease that a compatible guide nucleic acid (gNA), e.g., a gRNA, is capable of activating.
  • a composition provided herein further comprises a Cas protein that is related to the Cas nuclease that a compatible guide nucleic acid (gNA), e.g., a gRNA, is capable of activating.
  • a Cas protein comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the Cas nuclease amino acid sequence.
  • a Cas protein comprises a nuclease-inactive mutant of the Cas nuclease.
  • a Cas protein further comprises an effector domain.
  • a Cas protein lacks substantially all DNA cleavage activity.
  • Such a Cas protein can be generated, e.g., by introducing one or more mutations to an active Cas nuclease (e.g., a naturally occurring Cas nuclease).
  • a mutated Cas protein is considered to lack substantially all DNA cleavage activity when the DNA cleavage activity of the protein has no more than about 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less of the DNA cleavage activity of the corresponding non-mutated form, for example, nil or negligible as compared with the non- mutated form.
  • a Cas protein may comprise one or more mutations (e.g., a mutation in the RuvC domain of a type V-A Cas protein) and be used as a generic DNA binding protein with or without fusion to an effector domain.
  • Exemplary mutations include D908A, E993A, and D1263A with reference to the amino acid positions in AsCpf1; D832A, E925A, and D1180A with reference to the amino acid positions in LbCpf1; and D917A, E1006A, and D1255A with reference to the amino acid position numbering of the FnCpf1. More mutations can be designed and generated according to the crystal structure described in Yamano et al. (2016) CELL, 165: 949.
  • a Cas protein rather than losing nuclease activity to cleave all DNA, may lose the ability to cleave only the target strand or only the non-target strand of a double-stranded DNA, thereby being functional as a nickase (see, Gao et al. (2016) CELL RES., 26: 901). Accordingly, in certain embodiments, a Cas nuclease is a Cas nickase. In certain embodiments, a Cas nuclease has the activity to cleave the non-target strand but lacks substantially the activity to cleave the target strand, e.g., by a mutation in the Nuc domain.
  • a Cas nuclease has the cleavage activity to cleave the target strand but lacks substantially the activity to cleave the non-target strand. [0099] In certain embodiments, a Cas nuclease has the activity to cleave a double-stranded DNA and result in a double-strand break. [0100] Cas proteins that lack substantially all DNA cleavage activity or have the ability to cleave only one strand may also be identified from naturally occurring systems. For example, certain naturally occurring CRISPR-Cas systems may retain the ability to bind the target nucleotide sequence but lose entire or partial DNA cleavage activity in eukaryotic (e.g., mammalian or human) cells.
  • eukaryotic e.g., mammalian or human
  • Such type V-A proteins are disclosed, for example, in Kim et al. (2017) ACS SYNTH. BIOL.6(7): 1273-82 and Zhang et al. (2017) CELL DISCOV.3:17018.
  • the activity of a Cas protein e.g., Cas nuclease
  • altered activity of an engineered Cas protein comprises increased targeting efficiency and/or decreased off-target binding.
  • altered activity comprises modified binding, e.g., increased binding to the target locus (e.g., the target strand or the non-target strand) and/or decreased binding to off-target loci.
  • altered activity comprises altered charge in a region of the protein that associates with a single guide nucleic acid or dual guide nucleic acids.
  • altered activity of an engineered Cas protein comprises altered charge in a region of the protein that associates with the target strand and/or the non- target strand. In certain embodiments, altered activity of an engineered Cas protein comprises altered charge in a region of the protein that associates with an off-target locus.
  • the altered charge can include decreased positive charge, decreased negative charge, increased positive charge, or increased negative charge. For example, decreased negative charge and increased positive charge may generally strengthen binding to the nucleic acid(s) whereas decreased positive charge and increased negative charge may weaken binding to the nucleic acid(s).
  • altered activity comprises increased or decreased steric hindrance between the protein and a single guide nucleic acid or dual guide nucleic acids.
  • altered activity comprises increased or decreased steric hindrance between the protein and the target strand and/or the non-target strand. In certain embodiments, altered activity comprises increased or decreased steric hindrance between the protein and an off-target locus.
  • a modification or mutation comprises one or more substitutions of Lys, His, Arg, Glu, Asp, Ser, Gly, and/or Thr. In certain embodiments, a modification or mutation comprises one or more substitutions with Gly, Ala, Ile, Glu, and/or Asp. In certain embodiments, modification or mutation comprises one or more amino acid substitutions in the groove between the WED and RuvC domain of the Cas protein (e.g., a type V-A Cas protein).
  • altered activity of an engineered Cas protein comprises increased nuclease activity to cleave the target locus. In certain embodiments, altered activity of an engineered Cas protein comprises decreased nuclease activity to cleave an off-target locus. In certain embodiments, altered activity of an engineered Cas protein comprises altered helicase kinetics. In certain embodiments, an engineered Cas protein comprises a modification that alters formation of the CRISPR complex.
  • a protospacer adjacent motif (PAM) or PAM-like motif directs binding of a Cas protein complex to a target locus. Many Cas proteins have PAM specificity. The precise sequence and length requirements for the PAM differ depending on the Cas protein used.
  • PAM sequences are typically 2-5 base pairs in length and are adjacent to (but located on a different strand of target DNA from) the target nucleotide sequence. PAM sequences can be identified using any suitable method, such as testing cleavage, targeting, or modification of oligonucleotides having the target nucleotide sequence and different PAM sequences. [0104] Exemplary PAM sequences are provided in Tables 2 and 3.
  • a Cas protein comprises MAD7 and the PAM is TTTN, wherein N is A, C, G, or T.
  • a Cas protein comprises MAD7 and the PAM is CTTN, wherein N is A, C, G, or T.
  • a Cas protein comprises AsCpf1 and the PAM is TTTN, wherein N is A, C, G, or T.
  • a Cas protein comprises FnCpf1 and the PAM is 5' TTN, wherein N is A, C, G, or T.
  • PAM sequences for certain other type V-A Cas proteins are disclosed in Zetsche et al. (2015) CELL, 163: 759 and U.S. Patent No.9,982,279. Further, engineering of the PAM Interacting (PI) domain of a Cas protein may allow programing of PAM specificity, improve target site recognition fidelity, and/or increase the versatility of an engineered, non- naturally occurring system.
  • an engineered Cas protein comprises one or more nuclear localization signal (NLS) motifs.
  • an engineered Cas protein comprises at least 2 (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) NLS motifs.
  • Non-limiting examples of NLS motifs include: the NLS of SV40 large T-antigen, having the amino acid sequence of PKKKRKV (SEQ ID NO: 40); the NLS from nucleoplasmin, e.g., the nucleoplasmin bipartite NLS having the amino acid sequence of KRPAATKKAGQAKKKK (SEQ ID NO: 41); the C-myc NLS, having the amino acid sequence of PAAKRVKLD (SEQ ID NO: 42) or RQRRNELKRSP (SEQ ID NO: 43); the hRNPA1 M9 NLS, having the amino acid sequence of NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 44); the importin- ⁇ IBB domain NLS, having the amino acid sequence of RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 45); the myoma T protein NLS, having the amino acid sequence
  • the one or more NLS motifs are of sufficient strength to drive accumulation of the Cas protein in a detectable amount in the nucleus of a eukaryotic cell.
  • the strength of nuclear localization activity may derive from the number of NLS motif(s) in the Cas protein, the particular NLS motif(s) used, the position(s) of the NLS motif(s), or a combination of these and/or other factors.
  • an engineered Cas protein comprises at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) NLS motif(s) at or near the N-terminus (e.g., within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N-terminus).
  • an engineered Cas protein comprises at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) NLS motif(s) at or near the C- terminus (e.g., within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the C-terminus).
  • an engineered Cas protein comprises at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) NLS motif(s) at or near the C-terminus and at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) NLS motif(s) at or near the N-terminus.
  • the engineered Cas protein comprises one, two, or three NLS motifs at or near the C-terminus.
  • the engineered Cas protein comprises one NLS motif at or near the N-terminus and one, two, or three NLS motifs at or near the C-terminus. In certain embodiments, the engineered Cas protein comprises a nucleoplasmin NLS at or near the C-terminus.
  • Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to a nucleic acid-targeting protein, such that location within a cell may be visualized. Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting the protein, such as immunohistochemistry, Western blot, or enzyme activity assay.
  • Accumulation in the nucleus may also be determined indirectly, such as by an assay that detects the effect of the nuclear import of a Cas protein complex (e.g., assay for DNA cleavage or mutation at the target locus, or assay for altered gene expression activity) as compared to a control not exposed to the Cas protein or exposed to a Cas protein lacking one or more of the NLS motifs.
  • a Cas protein may comprise a chimeric Cas protein, e.g., a Cas protein having enhanced function by being a chimera. Chimeric Cas proteins may be new Cas proteins containing fragments from more than one naturally occurring Cas protein or variants thereof.
  • fragments of multiple type V-A Cas homologs may be fused to form a chimeric Cas protein.
  • a chimeric Cas protein comprises fragments of Cpf1 orthologs from multiple species and/or strains.
  • a Cas protein comprises one or more effector domains. The one or more effector domains may be located at or near the N-terminus of the Cas protein and/or at or near the C-terminus of the Cas protein.
  • an effector domain comprised in the Cas protein is a transcriptional activation domain (e.g., VP64), a transcriptional repression domain (e.g., a KRAB domain or an SID domain), an exogenous nuclease domain (e.g., FokI), a deaminase domain (e.g., cytidine deaminase or adenine deaminase), or a reverse transcriptase domain (e.g., a high fidelity reverse transcriptase domain).
  • a transcriptional activation domain e.g., VP64
  • a transcriptional repression domain e.g., a KRAB domain or an SID domain
  • an exogenous nuclease domain e.g., FokI
  • a deaminase domain e.g., cytidine deaminase or adenine deaminase
  • a Cas protein comprises one or more protein domains that enhance homology-directed repair (HDR) and/or inhibit non-homologous end joining (NHEJ).
  • HDR homology-directed repair
  • NHEJ non-homologous end joining
  • a Cas protein comprises a dominant negative version of p53-binding protein 1 (53BP1), for example, a fragment of 53BP1 comprising a minimum focus forming region (e.g., amino acids 1231-1644 of human 53BP1).
  • 53BP1 p53-binding protein 1
  • a Cas protein comprises a motif that is targeted by APC-Cdh1, such as amino acids 1-110 of human Geminin, thereby resulting in degradation of the fusion protein during the HDR non-permissive G1 phase of the cell cycle.
  • a Cas protein comprises an inducible or controllable domain.
  • inducers or controllers include light, hormones, and small molecule drugs.
  • a Cas protein comprises a light inducible or controllable domain.
  • a Cas protein comprises a chemically inducible or controllable domain.
  • a Cas protein comprises a tag protein or peptide for ease of tracking and/or purification.
  • tag proteins and peptides include fluorescent proteins (e.g., green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato), HIS tags (e.g., 6 ⁇ His tag, or gly-6xHis; 8xHis, or gly-8xHis), hemagglutinin (HA) tag, FLAG tag, 3xFLAG tag, and Myc tag.
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • CFP CFP
  • mCherry mCherry
  • tdTomato e.g., HIS tags
  • HIS tags e.g., 6 ⁇ His tag, or gly-6xHis; 8xHis, or gly-8xHis
  • HA hemagglutinin
  • FLAG tag FLAG tag
  • 3xFLAG tag 3xFLAG tag
  • a Cas protein is covalently conjugated to the non-protein moiety.
  • CRISPR-Associated protein Cas protein
  • Cas CRISPR-Associated nuclease
  • Cas nuclease CRISPR-Associated nuclease
  • a guide nucleic acid can be a single gNA (sgNA, e.g., sgRNA), in which the gNA is a single polynucleotide, or a dual gNA (e.g., dual gRNA), in which the gNA comprises two separate polynucleotides (these can in some cases be covalently linked, but not via a conventional internucleotide linkage).
  • a single guide nucleic acid is capable of activating a Cas nuclease alone (e.g., in the absence of a tracrRNA).
  • a gNA comprises a modulator nucleic acid and a targeter nucleic acid.
  • the modulator and targeter nucleic acids are part of a single polynucleotide.
  • the modulator and targeter nucleic acids are separate, e.g., not joined by a conventional nucleotide linkage, such as not joined at all.
  • the targeter nucleic acid comprises a spacer sequence and a targeter stem sequence.
  • the modulator nucleic acid comprises a modulator stem sequence and, generally, further nucleotides, such as nucleotides comprising a 5’ tail.
  • the modulator stem sequence and targeter stem sequence can each comprise any suitable number of nucleotides and are of sufficient complementarity that they can hybridize. In a single gNA there may be additional NTs between the targeter stem sequence and the modulator stem sequence; these can, in certain cases, form secondary structure, such as a loop.
  • the guide nucleic acid comprises a targeter nucleic acid that, in combination with a modulator nucleic acid, is capable of binding a Cas protein. In certain embodiments, the guide nucleic acid comprises a targeter nucleic acid that, in combination with a modulator nucleic acid, is capable of activating a Cas nuclease.
  • the system further comprises the Cas protein that the targeter nucleic acid and the modulator nucleic acid are capable of binding or the Cas nuclease that the targeter nucleic acid and the modulator nucleic acid are capable of activating.
  • the single or dual guide nucleic acids need to be the compatible with a Cas protein (e.g., Cas nuclease) to provide an operative CRISPR system.
  • the targeter stem sequence and the modulator stem sequence can be derived from a naturally occurring crRNA capable of activating a Cas nuclease in the absence of a tracrRNA.
  • the targeter stem sequence and the modulator stem sequence can be derived from a naturally occurring set of crRNA and tracrRNA, respectively, that are capable of activating a Cas nuclease.
  • the nucleotide sequences of the targeter stem sequence and the modulator stem sequence are identical to the corresponding stem sequences of a stem-loop structure in such naturally occurring crRNA.
  • Guide nucleic acid sequences that are operative with a type II or type V Cas protein are known in the art and are disclosed, for example, in U.S. Patent Nos.9,790,490, 9,896,696, 10,113,179, and 10,266,850, and U.S. Patent Application Publication No.2014/0242664. It is understood that these sequences are merely illustrative, and other guide nucleic acid sequences may also be used with these Cas proteins. TABLE 4: Type V-A Cas Protein and Corresponding Single Guide Nucleic Acid Sequences
  • the modulator sequence in the scaffold sequence is underlined; the targeter stem sequence in the scaffold sequence is bold-underlined. It is understood that a “scaffold sequence” listed herein constitutes a portion of a single guide nucleic acid. Additional nucleotide sequences, other than the spacer sequence, can be comprised in the single guide nucleic acid. 2 In the consensus PAM sequences, N represents A, C, G, or T. Where the PAM sequence is preceded by “5’,” it means that the PAM is located immediately upstream of the target nucleotide sequence when using the non-target strand (i.e., the strand not hybridized with the spacer sequence) as the coordinate. TABLE 5: Type V-A Cas Protein and Corresponding Dual Guide Nucleic Acid Sequences
  • a “modulator sequence” listed herein may constitute the nucleotide sequence of a modulator nucleic acid.
  • additional nucleotide sequences can be comprised in the modulator nucleic acid 5’ and/or 3’ to a “modulator sequence” listed herein.
  • N represents A, C, G, or T.
  • the PAM sequence is preceded by “5’,” it means that the PAM is located immediately upstream of the target nucleotide sequence when using the non-target strand (i.e., the strand not hybridized with the spacer sequence) as the coordinate.
  • a guide nucleic acid in the context of a type V-A CRISPR- Cas system, comprises a targeter stem sequence listed in Table 5. The same targeter stem sequences, as a portion of scaffold sequences, are bold-underlined in Table 4. [0121]
  • a guide nucleic acid is a single guide nucleic acid that comprises, from 5’ to 3’, a modulator stem sequence, a loop sequence, a targeter stem sequence, and a spacer sequence.
  • the targeter stem sequence in the single guide nucleic acid is listed in Table 4 as a bold-underlined portion of scaffold sequence, and the modulator stem sequence is complementary (e.g., 100% complementary) to the targeter stem sequence.
  • the single guide nucleic acid comprises, from 5’ to 3’, a modulator sequence listed in Table 4 as an underlined portion of a scaffold sequence, a loop sequence, a targeter stem sequence a bold-underlined portion of the same scaffold sequence, and a spacer sequence.
  • an engineered, non-naturally occurring system comprises a single guide nucleic acid comprising a scaffold sequence listed in Table 4.
  • the system further comprises a Cas protein (e.g., Cas nuclease) comprising an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in the SEQ ID NO listed in the same line of Table 4.
  • the system further comprises a Cas protein (e.g., Cas nuclease) comprising the amino acid sequence set forth in the SEQ ID NO listed in the same line of Table 4.
  • the system is useful for targeting, editing, or modifying a nucleic acid comprising a target nucleotide sequence close or adjacent to (e.g., immediately downstream of) a PAM listed in the same line of Table 4 when using the non-target strand (i.e., the strand not hybridized with the spacer sequence) as the coordinate.
  • a guide nucleic acid e.g., dual gNA, comprises a targeter guide nucleic acid that comprises, from 5’ to 3’, a targeter stem sequence and a spacer sequence.
  • the targeter stem sequence in the targeter nucleic acid is listed in Table 5.
  • an engineered, non-naturally occurring system comprises the targeter nucleic acid and a modulator stem sequence complementary (e.g., 100% complementary) to the targeter stem sequence.
  • the modulator nucleic acid comprises a modulator sequence listed in the same line of Table 5.
  • the system further comprises a Cas protein (e.g., Cas nuclease) comprising an amino acid sequence at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in the SEQ ID NO listed in the same line of Table 5.
  • the system further comprises a Cas protein (e.g., Cas nuclease) comprising the amino acid sequence set forth in the SEQ ID NO listed in the same line of Table 5.
  • a Cas protein e.g., Cas nuclease
  • the system is useful for targeting, editing, or modifying a nucleic acid comprising a target nucleotide sequence close or adjacent to (e.g., immediately downstream of) a PAM listed in the same line of Table 5 when using the non-target strand (i.e., the strand not hybridized with the spacer sequence) as the coordinate.
  • a single guide nucleic acid, the targeter nucleic acid, and/or the modulator nucleic acid can be synthesized chemically or produced in a biological process (e.g., catalyzed by an RNA polymerase in an in vitro reaction). Such reaction or process may limit the lengths of the single guide nucleic acid, targeter nucleic acid, and/or modulator nucleic acid.
  • a single guide nucleic acid is no more than 100, 90, 80, 70, 60, 50, 40, 30, or 25 nucleotides in length.
  • a single guide nucleic acid is at least 20, 25, 30, 40, 50, 60, 70, 80, or 90 nucleotides in length.
  • the single guide nucleic acid is 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 20-25, 25-100, 25-90, 25-80, 25-70, 25-60, 25-50, 25-40, 25-30, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50-60, 60-100, 60-90, 60-80, 60-70, 70-100, 70-90, 70-80, 80-100, 80-90, or 90-100 nucleotides in length.
  • a targeter nucleic acid is no more than 100, 90, 80, 70, 60, 50, 40, 30, or 25 nucleotides in length. In certain embodiments, a targeter nucleic acid is at least 20, 25, 30, 40, 50, 60, 70, 80, or 90 nucleotides in length.
  • the targeter nucleic acid is 20- 100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 20-25, 25-100, 25-90, 25-80, 25-70, 25- 60, 25-50, 25-40, 25-30, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-100, 40-90, 40- 80, 40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50-60, 60-100, 60-90, 60-80, 60-70, 70- 100, 70-90, 70-80, 80-100, 80-90, or 90-100 nucleotides in length.
  • a modulator nucleic acid is no more than 100, 90, 80, 70, 60, 50, 40, 30, or 20 nucleotides in length. In certain embodiments, a modulator nucleic acid is at least 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, or 90 nucleotides in length.
  • the targeter stem sequence and the modulator stem sequence each consist of 4, 5, 6, 7, 8, 9, or 10 nucleotides. It is understood that the composition of the nucleotides in each sequence affects the stability of the duplex, and a C-G base pair confers greater stability than an A-U base pair.
  • 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-80%, 30%-70%, 30%-60%, 30%- 50%, 30%-40%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-80%, 50%-70%, 50%-60%, 60%-80%, 60%-70%, or 70%-80% of the base pairs are C-G base pairs.
  • the targeter stem sequence and the modulator stem sequence each consist of 5 nucleotides. As such, the targeter stem sequence and the modulator stem sequence form a duplex of 5 base pairs. In certain embodiments, 0-4, 0-3, 0-2, 0-1, 1-5, 1-4, 1-3, 1-2, 2-5, 2-4, 2-3, 3-5, 3-4, or 4-5 out of the 5 base pairs are C-G base pairs. In certain embodiments, 0, 1, 2, 3, 4, or 5 out of the 5 base pairs are C-G base pairs. In certain embodiments, the targeter stem sequence consists of 5’-GUAGA-3’ and the modulator stem sequence consists of 5’-UCUAC-3’.
  • the targeter nucleic acid does not comprise any additional nucleotide 5’ to the targeter stem sequence.
  • the targeter nucleic acid or the single guide nucleic acid further comprises an additional nucleotide sequence containing one or more nucleotides at the 3’ end that does not hybridize with the target nucleotide sequence.
  • the additional nucleotide sequence may protect the targeter nucleic acid from degradation by 3’-5’ exonuclease.
  • the additional nucleotide sequence is no more than 100 nucleotides in length.
  • Such secondary structure may increase the specificity of guide nucleic acid or the engineered, non-naturally occurring system (see, Kocak et al. (2019) Nat. Biotech.37: 657- 66).
  • the free energy change during the hairpin formation is greater than or equal to -20 kcal/mol, -15 kcal/mol, -14 kcal/mol, -13 kcal/mol, -12 kcal/mol, -11 kcal/mol, or -10 kcal/mol.
  • the free energy change during the hairpin formation is greater than or equal to -5 kcal/mol, -6 kcal/mol, -7 kcal/mol, -8 kcal/mol, -9 kcal/mol, -10 kcal/mol, -11 kcal/mol, -12 kcal/mol, -13 kcal/mol, -14 kcal/mol, or -15 kcal/mol.
  • the targeter nucleic acid or the single guide nucleic acid does not comprise any nucleotide 3’ to the spacer sequence.
  • the modulator nucleic acid further comprises an additional nucleotide sequence 3’ to the modulator stem sequence.
  • the additional nucleotide sequence comprises at least 1 (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50) nucleotides.
  • the additional nucleotide sequence consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides. In certain embodiments, the additional nucleotide sequence consists of 1 nucleotide (e.g., uridine). In certain embodiments, the additional nucleotide sequence consists of 2 nucleotides. In certain embodiments, the additional nucleotide sequence is reminiscent to the loop or a fragment thereof (e.g., one, two, three, or four nucleotides at the 5’ end of the loop) in a crRNA of a corresponding single guide CRISPR-Cas system.
  • an additional nucleotide sequence 3’ to the modulator stem sequence can be dispensable. Accordingly, in certain embodiments, the modulator nucleic acid does not comprise any additional nucleotide 3’ to the modulator stem sequence. [0131] It is understood that the additional nucleotide sequence 5’ to the targeter stem sequence and the additional nucleotide sequence 3’ to the modulator stem sequence, if present, may interact with each other.
  • nucleotide immediately 5’ to the targeter stem sequence and the nucleotide immediately 3’ to the modulator stem sequence do not form a Watson-Crick base pair (otherwise they would constitute part of the targeter stem sequence and part of the modulator stem sequence, respectively), other nucleotides in the additional nucleotide sequence 5’ to the targeter stem sequence and the additional nucleotide sequence 3’ to the modulator stem sequence may form one, two, three, or more base pairs (e.g., Watson-Crick base pairs). Such interaction may affect the stability of a complex comprising the targeter nucleic acid and the modulator nucleic acid.
  • the stability of a complex comprising a targeter nucleic acid and a modulator nucleic acid can be assessed by the Gibbs free energy change ( ⁇ G) during the formation of the complex, either calculated or actually measured.
  • ⁇ G Gibbs free energy change
  • the ⁇ G during the formation of the complex correlates generally with the ⁇ G during the formation of a secondary structure within the corresponding single guide nucleic acid.
  • RNAfold (rna.tbi.univie.ac.at/cgi- bin/RNAWebSuite/RNAfold.cgi) as disclosed in Gruber et al. (2008) Nucleic Acids Res., 36(Web Server issue): W70–W74. Unless indicated otherwise, the ⁇ G values in the present disclosure are calculated by RNAfold for the formation of a secondary structure within a corresponding single guide nucleic acid.
  • the ⁇ G is lower than or equal to -1 kcal/mol, e.g., lower than or equal to -2 kcal/mol, lower than or equal to -3 kcal/mol, lower than or equal to -4 kcal/mol, lower than or equal to -5 kcal/mol, lower than or equal to -6 kcal/mol, lower than or equal to -7 kcal/mol, lower than or equal to -7.5 kcal/mol, or lower than or equal to -8 kcal/mol.
  • the ⁇ G is greater than or equal to -10 kcal/mol, e.g., greater than or equal to -9 kcal/mol, greater than or equal to -8.5 kcal/mol, or greater than or equal to -8 kcal/mol. In certain embodiments, the ⁇ G is in the range of -10 to -4 kcal/mol.
  • the ⁇ G is in the range of -8 to -4 kcal/mol, -7 to -4 kcal/mol, -6 to -4 kcal/mol, -5 to -4 kcal/mol, -8 to -4.5 kcal/mol, -7 to -4.5 kcal/mol, -6 to -4.5 kcal/mol, or -5 to - 4.5 kcal/mol.
  • the ⁇ G is about -8 kcal/mol, -7 kcal/mol, -6 kcal/mol, -5 kcal/mol, -4.9 kcal/mol, -4.8 kcal/mol, -4.7 kcal/mol, -4.6 kcal/mol, -4.5 kcal/mol, -4.4 kcal/mol, -4.3 kcal/mol, -4.2 kcal/mol, -4.1 kcal/mol, or -4 kcal/mol.
  • the ⁇ G may be affected by a sequence in the targeter nucleic acid that is not within the targeter stem sequence, and/or a sequence in the modulator nucleic acid that is not within the modulator stem sequence.
  • one or more base pairs e.g., Watson- Crick base pair
  • Watson- Crick base pair may reduce the ⁇ G, i.e., stabilize the nucleic acid complex.
  • the nucleotide immediately 5’ to the targeter stem sequence comprises a uracil or is a uridine
  • the nucleotide immediately 3’ to the modulator stem sequence comprises a uracil or is a uridine, thereby forming a nonconventional U-U base pair.
  • the modulator nucleic acid or the single guide nucleic acid comprises a nucleotide sequence referred to herein as a “5’ tail” positioned 5’ to the modulator stem sequence.
  • the 5’ tail is a nucleotide sequence positioned 5’ to the stem-loop structure of the crRNA.
  • a 5’ tail in an engineered type V-A CRISPR-Cas system can be reminiscent to the 5’ tail in a corresponding naturally occurring type V-A CRISPR-Cas system.
  • the 5’ tail may participate in the formation of the CRISPR-Cas complex.
  • the 5’ tail forms a pseudoknot structure with the modulator stem sequence, which is recognized by the Cas protein (see, Yamano et al. (2016) Cell, 165: 949).
  • the 5’ tail is at least 3 (e.g., at least 4 or at least 5) nucleotides in length.
  • the modulator nucleic acid comprises a uridine or a uracil-containing nucleotide 5’ to the modulator stem sequence.
  • the 5’ tail comprises the nucleotide sequence of 5’- AUU-3’.
  • the 5’ tail comprises the nucleotide sequence of 5’-AAUU-3’.
  • the 5’ tail comprises the nucleotide sequence of 5’-UAAUU-3’.
  • the 5’ tail is positioned immediately 5’ to the modulator stem sequence.
  • the single guide nucleic acid, the targeter nucleic acid, and/or the modulator nucleic acid are designed to reduce the degree of secondary structure other than the hybridization between the targeter stem sequence and the modulator stem sequence. In certain embodiments, no more than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the single guide nucleic acid other than the targeter stem sequence and the modulator stem sequence participate in self-complementary base pairing when optimally folded.
  • nucleotides of the targeter nucleic acid and/or the modulator nucleic acid participate in self-complementary base pairing when optimally folded.
  • Optimal folding may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res.9 (1981), 133-148).
  • Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g., A. R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62).
  • the targeter nucleic acid is directed to a specific target nucleotide sequence, and a donor template can be designed to modify the target nucleotide sequence or a sequence nearby. It is understood, therefore, that association of the single guide nucleic acid, the targeter nucleic acid, or the modulator nucleic acid with a donor template can increase editing efficiency and reduce off-targeting.
  • the single guide nucleic acid or the modulator nucleic acid further comprises a donor template-recruiting sequence capable of hybridizing with a donor template (see Figure 2B).
  • Donor templates are described in the “Donor Templates” subsection of section II infra.
  • the donor template and donor template-recruiting sequence can be designed such that they bear sequence complementarity.
  • the donor template-recruiting sequence is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) complementary to at least a portion of the donor template.
  • the donor template-recruiting sequence is 100% complementary to at least a portion of the donor template.
  • the donor template comprises an engineered sequence not homologous to the sequence to be repaired, the donor template-recruiting sequence is capable of hybridizing with the engineered sequence in the donor template.
  • the donor template-recruiting sequence is at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides in length.
  • the donor template-recruiting sequence is positioned at or near the 5’ end of the single guide nucleic acid or at or near the 5’ end of the modulator nucleic acid.
  • the donor template-recruiting sequence is linked to the 5’ tail, if present, or to the modulator stem sequence, of the single guide nucleic acid or the modulator nucleic acid through an internucleotide bond or a nucleotide linker.
  • the single guide nucleic acid or the modulator nucleic acid further comprises an editing enhancer sequence, which increases the efficiency of gene editing and/or homology-directed repair (HDR) (see Figure 2C). Exemplary editing enhancer sequences are described in Park et al. (2016) Nat. Commun.9: 3313.
  • the editing enhancer sequence is positioned 5’ to the 5’ tail, if present, or 5’ to the single guide nucleic acid or the modulator stem sequence.
  • the editing enhancer sequence is 1-50, 4-50, 9-50, 15-50, 25-50, 1-25, 4-25, 9-25, 15-25, 1-15, 4-15, 9-15, 1-9, 4-9, or 1-4 nucleotides in length.
  • the editing enhancer sequence is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55 nucleotides in length.
  • the editing enhancer sequence is designed to minimize homology to the target nucleotide sequence or any other sequence that the engineered, non-naturally occurring system may be contacted to, e.g., the genome sequence of a cell into which the engineered, non-naturally occurring system is delivered.
  • the editing enhancer is designed to minimize the presence of hairpin structure.
  • the editing enhancer can comprise one or more of the chemical modifications disclosed herein.
  • the single guide nucleic acid, the modulator nucleic acid, and/or the targeter nucleic acid can further comprise a protective nucleotide sequence that prevents or reduces nucleic acid degradation.
  • the protective nucleotide sequence is at least 5 (e.g., at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50) nucleotides in length.
  • the length of the protective nucleotide sequence increases the time for an exonuclease to reach the 5’ tail, modulator stem sequence, targeter stem sequence, and/or spacer sequence, thereby protecting these portions of the single guide nucleic acid, the modulator nucleic acid, and/or the targeter nucleic acid from degradation by an exonuclease.
  • the protective nucleotide sequence forms a secondary structure, such as a hairpin or a tRNA structure, to reduce the speed of degradation by an exonuclease (see, for example, Wu et al. (2016) Cell. Mol. Life Sci., 75(19): 3593-3607).
  • Secondary structures can be predicted by methods known in the art, such as the online webserver RNAfold developed at University of Vienna using the centroid structure prediction algorithm (see, Gruber et al. (2008) Nucleic Acids Res., 36: W70).
  • Certain chemical modifications, which may be present in the protective nucleotide sequence can also prevent or reduce nucleic acid degradation, as disclosed in the “RNA Modifications” subsection infra.
  • the targeter nucleic acid comprises a protective nucleotide sequence at the 5’ end, at the 3’ end, or at both ends, optionally through a nucleotide linker.
  • various nucleotide sequences can be present in the 5’ portion of a single nucleic acid or a modulator nucleic acid, including but not limited to a donor template- recruiting sequence, an editing enhancer sequence, a protective nucleotide sequence, and a linker connecting such sequence to the 5’ tail, if present, or to the modulator stem sequence.
  • the single guide nucleic acid or the modulator nucleic acid comprises a nucleotide sequence that is both a donor template-recruiting sequence and an editing enhancer sequence.
  • the single guide nucleic acid or the modulator nucleic acid comprises a nucleotide sequence that is both a donor template-recruiting sequence and a protective sequence.
  • the single guide nucleic acid or the modulator nucleic acid comprises a nucleotide sequence that is both an editing enhancer sequence and a protective sequence.
  • the single guide nucleic acid or the modulator nucleic acid comprises a nucleotide sequence that is a donor template-recruiting sequence, an editing enhancer sequence, and a protective sequence.
  • the nucleotide sequence 5’ to the 5’ tail, if present, or 5’ to the modulator stem sequence is 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-90, 30-80, 30- 70, 30-60, 30-50, 30-40, 40-90, 40-80, 40-70, 40-60, 40-50, 50-90, 50-80, 50-70, 50-60, 60-90, 60-80, 60-70, 70-90, 70-80, or 80-90 nucleotides in length.
  • an engineered, non-naturally occurring system further comprises one or more compounds (e.g., small molecule compounds) that enhance HDR and/or inhibit NHEJ.
  • compounds e.g., small molecule compounds
  • Exemplary compounds having such functions are described in Maruyama et al. (2015) Nat Biotechnol.33(5): 538-42; Chu et al. (2015) Nat Biotechnol.33(5): 543-48; Yu et al. (2015) Cell Stem Cell 16(2): 142-47; Pinder et al. (2015) Nucleic Acids Res.43(19): 9379-92; and Yagiz et al. (2019) Commun. Biol.2: 198.
  • an engineered, non- naturally occurring system further comprises one or more compounds selected from the group consisting of DNA ligase IV antagonists (e.g., SCR7 compound, Ad4 E1B55K protein, and Ad4 E4orf6 protein), RAD51 agonists (e.g., RS-1), DNA-dependent protein kinase (DNA-PK) antagonists (e.g., NU7441 and KU0060648), ⁇ 3-adrenergic receptor agonists (e.g., L755507), inhibitors of intracellular protein transport from the ER to the Golgi apparatus (e.g., brefeldin A), and any combinations thereof.
  • DNA ligase IV antagonists e.g., SCR7 compound, Ad4 E1B55K protein, and Ad4 E4orf6 protein
  • RAD51 agonists e.g., RS-1
  • DNA-PK DNA-dependent protein kinase
  • ⁇ 3-adrenergic receptor agonists
  • an engineered, non-naturally occurring system comprising a targeter nucleic acid and a modulator nucleic acid is tunable or inducible.
  • the targeter nucleic acid, the modulator nucleic acid, and/or the Cas protein can be introduced to the target nucleotide sequence at different times, the system becoming active only when all components are present.
  • the amounts of the targeter nucleic acid, the modulator nucleic acid, and/or the Cas protein can be titrated to achieve desired efficiency and specificity.
  • Guide nucleic acids including a single guide nucleic acid, a targeter nucleic acid, and/or a modulator nucleic acid, may comprise a DNA (e.g., modified DNA), an RNA (e.g., modified RNA), or a combination thereof.
  • the single guide nucleic acid comprises a DNA (e.g., modified DNA), an RNA (e.g., modified RNA), or a combination thereof.
  • the targeter nucleic acid comprises a DNA (e.g., modified DNA), an RNA (e.g., modified RNA), or a combination thereof.
  • the modulator nucleic acid comprises a DNA (e.g., modified DNA), an RNA (e.g., modified RNA), or a combination thereof.
  • Spacer sequences can be presented as DNA sequences by including thymidines (T) rather than uridines (U). It is understood that corresponding RNA sequences and DNA/RNA chimeric sequences are also contemplated. For example, where the spacer sequence is an RNA, its sequence can be derived from a DNA sequence disclosed herein by replacing each T with U.
  • engineered, non-naturally occurring systems comprising a targeter nucleic acid comprising: a spacer sequence designed to hybridize with a target nucleotide sequence and a targeter stem sequence; and a modulator nucleic acid comprising a modulator stem sequence complementary to the targeter stem sequence, and, optionally, a 5’ sequence, e.g., a tail sequence, wherein, in a single guide nucleic acid the targeter nucleic acid and the modulator nucleic acid are part of a single polynucleotide, and in a dual guide nucleic acid, the targeter nucleic acid and the modulator nucleic acid are separate nucleic acids; modifications can include one or more chemical modifications to one or more nucleotides or internucleotide linkages at or near the 3’ end of the targeter nucleic acid (dual and single and the targeter nucleic acid
  • the Cas nuclease is a type V-A Cas nuclease.
  • Modulator and/or targeter nucleic sequences can include further sequences, as detailed in the Guide Nucleic Acids section, and modifications can be in these further sequences, as appropriate and apparent to one of skill in the art.
  • the modulator nucleic acid may comprise a DNA (e.g., modified DNA), an RNA (e.g., modified RNA), or a combination thereof.
  • the targeter nucleic acid is an RNA and the modulator nucleic acid is an RNA.
  • a targeter nucleic acid in the form of an RNA is also called targeter RNA, and a modulator nucleic acid in the form of an RNA is also called modulator RNA.
  • the nucleotide sequences disclosed herein are presented as DNA sequences by including thymidines (T) and/or RNA sequences including uridines (U). It is understood that corresponding DNA sequences, RNA sequences, and DNA/RNA chimeric sequences are also contemplated.
  • RNA e.g., a gRNA.
  • 5-100%, 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 95-100%, 99-100%, 99.5-100% of the gNA is gRNA.
  • 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-80%, 30%-70%, 30%-60%, 30%-50%, 30%-40%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%- 80%, 50%-70%, 50%-60%, 60%-80%, 60%-70%, or 70%-80% of gNA is RNA.
  • 50% of the gNA is RNA.
  • 70% of the gNA is RNA.
  • 90% of the gNA is RNA.
  • 100% of the gNA is RNA, e.g., a gRNA.
  • the remaining portion of the gNA that is not RNA comprises a modified ribonucleotide, a deoxyribonucleotide, a modified deoxyribonucleotide, or a synthetic, e.g., unnatural nucleotide, for example, not intended to be limiting, threose nucleic acid, locked nucleic acid, peptide nucleic acid, arabinonucleic acid, hexose nucleic acid, among others.
  • the targeter nucleic acid and/or the modulator nucleic acid are RNAs with one or more modifications in a ribose group, one or more modifications in a phosphate group, one or more modifications in a nucleobase, one or more terminal modifications, or a combination thereof.
  • Exemplary modifications are disclosed in U.S. Patent Nos.10,900,034 and 10,767,175, U.S. Patent Application Publication No.2018/0119140, Watts et al. (2008) Drug Discov. Today 13: 842-55, and Hendel et al. (2015) NAT. BIOTECHNOL.33: 985.
  • a targeter nucleic acid e.g., RNA
  • the 3’ end of the targeter nucleic acid comprises the spacer sequence.
  • the 3’ end of the targeter nucleic acid comprises the targeter stem sequence. Exemplary modifications are disclosed in Dang et al. (2015) Genome Biol.16: 280, Kocaz et al. (2019) Nature Biotech.37: 657-66, Liu et al.
  • Modifications in a ribose group include but are not limited to modifications at the 2′ position or modifications at the 4′ position.
  • the ribose comprises 2′-O-C1-4alkyl, such as 2′-O-methyl (2′-OMe, or M).
  • the ribose comprises 2′-O-C1-3alkyl-O-C1-3alkyl, such as 2′-methoxyethoxy (2′-O—CH 2 CH 2 OCH 3 ) also known as 2′-O-(2-methoxyethyl) or 2′-MOE.
  • the ribose comprises 2′-O-allyl.
  • the ribose comprises 2′-O-2,4-Dinitrophenol (DNP).
  • the ribose comprises 2′-halo, such as 2′-F, 2′-Br, 2′-Cl, or 2′-I.
  • the ribose comprises 2′-NH 2 .
  • the ribose comprises 2′-H (e.g., a deoxynucleotide). In certain embodiments, the ribose comprises 2′-arabino or 2′-F- arabino. In certain embodiments, the ribose comprises 2′-LNA or 2′-ULNA. In certain embodiments, the ribose comprises a 4′-thioribosyl. [0151] Modifications can also include a deoxy group, for example a 2′-deoxy-3′- phosphonoacetate (DP), a 2′-deoxy-3′-thiophosphonoacetate (DSP).
  • DP 2′-deoxy-3′- phosphonoacetate
  • DSP 2′-deoxy-3′-thiophosphonoacetate
  • Internucleotide linkage modifications in a phosphate group include but are not limited to a phosphorothioate (S), a chiral phosphorothioate, a phosphorodithioate, a boranophosphonate, a C 1-4 alkyl phosphonate such as a methylphosphonate, a boranophosphonate, a phosphonocarboxylate such as a phosphonoacetate (P), a phosphonocarboxylate ester such as a phosphonoacetate ester, an amide, a thiophosphonocarboxylate such as a thiophosphonoacetate (SP), a thiophosphonocarboxylate ester such as a thiophosphonoacetate ester, and a 2′,5′-linkage having a phosphodiester or any of the modified phosphates above.
  • S phosphorothioate
  • nucleobase examples include but are not limited to 2-thiouracil, 2- thiocytosine, 4-thiouracil, 6-thioguanine, 2-aminoadenine, 2-aminopurine, pseudouracil, hypoxanthine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5- methylcytosine, 5-methyluracil, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5,6- dehydrouracil, 5-propynylcytosine, 5-propynyluracil, 5-ethynylcytosine, 5-ethynyluracil, 5- allyluracil, 5-allylcytosine, 5-aminoallyluracil, 5-aminoallyl-cytosine, 5-bromouracil, 5- iodouraci
  • Terminal modifications include but are not limited to polyethylene glycol (PEG), hydrocarbon linkers (such as heteroatom (O,S,N)-substituted hydrocarbon spacers; halo- substituted hydrocarbon spacers; keto-, carboxyl-, amido-, thionyl-, carbamoyl-, thionocarbamaoyl-containing hydrocarbon spacers, propanediol), spermine linkers, dyes such as fluorescent dyes (for example, fluoresceins, rhodamines, cyanines), quenchers (for example, dabcyl, BHQ), and other labels (for example biotin, digoxigenin, acridine, streptavidin, avidin, peptide
  • PEG polyethylene glycol
  • hydrocarbon linkers such as heteroatom (O,S,N)-substituted hydrocarbon spacers
  • halo- substituted hydrocarbon spacers keto-, carboxyl-, amido-, thiony
  • a terminal modification comprises a conjugation (or ligation) of the RNA to another molecule comprising an oligonucleotide (such as deoxyribonucleotides and/or ribonucleotides), a peptide, a protein, a sugar, an oligosaccharide, a steroid, a lipid, a folic acid, a vitamin and/or other molecule.
  • an oligonucleotide such as deoxyribonucleotides and/or ribonucleotides
  • a terminal modification incorporated into the RNA is located internally in the RNA sequence via a linker such as 2-(4-butylamidofluorescein)propane-1,3-diol bis(phosphodiester) linker, which is incorporated as a phosphodiester linkage and can be incorporated anywhere between two nucleotides in the RNA.
  • a linker such as 2-(4-butylamidofluorescein)propane-1,3-diol bis(phosphodiester) linker, which is incorporated as a phosphodiester linkage and can be incorporated anywhere between two nucleotides in the RNA.
  • modifications can include 2′-O-methyl (M), a phosphorothioate (S), a phosphonoacetate (P), a thiophosphonoacetate (SP), a 2′-O-methyl-3′- phosphorothioate (MS), a 2′-O-methyl-3′-phosphonoacetate (MP), a 2′-O-methyl-3′- thiophosphonoacetate (MSP), a 2′-deoxy-3′-phosphonoacetate (DP), a 2′-deoxy-3′- thiophosphonoacetate (DSP), or a combination thereof, at or near either the 3’ or 5’ end of either the targeter or modulator nucleic acid, as appropriate for single or dual gNA.
  • M 2′-O-methyl
  • S phosphorothioate
  • P phosphonoacetate
  • SP thiophosphonoacetate
  • MS 2′-O-methyl-3′- phosphorothioate
  • MS 2′
  • modifications can include either a 5’ or a 3’ propanediol or C3 linker modification.
  • the modification alters the stability of the RNA.
  • the modification enhances the stability of the RNA, e.g., by increasing nuclease resistance of the RNA relative to a corresponding RNA without the modification.
  • Stability- enhancing modifications include but are not limited to incorporation of 2′-O-methyl, a 2′-O-C 1- 4 alkyl, 2′-halo (e.g., 2′-F, 2′-Br, 2′-Cl, or 2′-I), 2′MOE, a 2′-O-C 1-3 alkyl-O-C 1-3 alkyl, 2′-NH 2 , 2′-H (or 2′-deoxy), 2′-arabino, 2′-F-arabino, 4′-thioribosyl sugar moiety, 3′-phosphorothioate, 3′- phosphonoacetate, 3′-thiophosphonoacetate, 3′-methylphosphonate, 3′-boranophosphate, 3′- phosphorodithioate, locked nucleic acid (“LNA”) nucleotide which comprises a methylene bridge between the 2' and 4' carbons of the ribose ring, and unlocked nucleic acid
  • modifications are suitable for use as a protecting group to prevent or reduce degradation of the 5’ sequence, e.g., a tail sequence, modulator stem sequence (dual guide nucleic acids), targeter stem sequence (dual guide nucleic acids), and/or spacer sequence (see, the “Targeter and Modulator nucleic acids” subsection).
  • the modification alters the specificity of the engineered, non- naturally occurring system.
  • the modification enhances the specificity of the engineered, non-naturally occurring system, e.g., by enhancing on-target binding and/or cleavage, or reducing off-target binding and/or cleavage, or a combination thereof.
  • Specificity- enhancing modifications include but are not limited to 2-thiouracil, 2-thiocytosine, 4-thiouracil, 6-thioguanine, 2-aminoadenine, and pseudouracil.
  • the modification alters the immunostimulatory effect of the RNA relative to a corresponding RNA without the modification.
  • the modification reduces the ability of the RNA to activate TLR7, TLR8, TLR9, TLR3, RIG-I, and/or MDA5.
  • the targeter nucleic acid and/or the modulator nucleic acid comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 modified nucleotides or internucleotide linkages.
  • the modification can be made at one or more positions in the targeter nucleic acid and/or the modulator nucleic acid such that these nucleic acids retain functionality.
  • the modified nucleic acids can still direct the Cas protein to the target nucleotide sequence and allow the Cas protein to exert its effector function.
  • the particular modification(s) at a position may be selected based on the functionality of the nucleotide or internucleotide linkage at the position.
  • a specificity-enhancing modification may be suitable for a nucleotide or internucleotide linkage in the spacer sequence, the targeter stem sequence, or the modulator stem sequence.
  • a stability-enhancing modification may be suitable for one or more terminal nucleotides or internucleotide linkages in the targeter nucleic acid and/or the modulator nucleic acid.
  • At least 1 e.g., at least 2, at least 3, at least 4, or at least 5 terminal nucleotides or internucleotide linkages at or near the 5’ end and/or at least 1 (e.g., at least 2, at least 3, at least 4, or at least 5) terminal nucleotides or internucleotide linkages at or near the 3’ end of the targeter nucleic acid are modified.
  • At least 1 e.g., at least 2, at least 3, at least 4, or at least 5 terminal nucleotides or internucleotide linkages at or near the 5’ end and/or at least 1 (e.g., at least 2, at least 3, at least 4, or at least 5) terminal nucleotides or internucleotide linkages at or near the 3’ end of the modulator nucleic acid are modified.
  • the targeter or modulator nucleic acid is a combination of DNA and RNA
  • the nucleic acid as a whole is considered as an RNA
  • the DNA nucleotide(s) are considered as modification(s) of the RNA, including a 2′-H modification of the ribose and optionally a modification of the nucleobase.
  • the targeter nucleic acid and the modulator nucleic acid while not in the same nucleic acids, i.e., not linked end-to-end through a traditional internucleotide bond, can be covalently conjugated to each other through one or more chemical modifications introduced into these nucleic acids, thereby increasing the stability of the double-stranded complex and/or improving other characteristics of the system.
  • the targeter nucleic acid and the modulator nucleic acid while not in the same nucleic acids, i.e., not linked end-to-end through a traditional internucleotide bond, can be covalently conjugated to each other through one or more chemical modifications introduced into these nucleic acids, thereby increasing the stability of the double-stranded complex and/or improving other characteristics of the system.
  • composition and methods for targeting, editing, and/or modifying genomic DNA can be useful for targeting, editing, and/or modifying a target nucleic acid, such as a DNA (e.g., genomic DNA) in a cell or organism.
  • a target nucleic acid such as a DNA (e.g., genomic DNA) in a cell or organism.
  • the present invention provides a method of cleaving a target nucleic acid (e.g., DNA) comprising the sequence of a preselected target sequence or a portion thereof, the method comprising contacting the target DNA with an engineered, non-naturally occurring system disclosed herein, thereby resulting in cleavage of the target DNA.
  • the present invention provides a method of binding a target nucleic acid (e.g., DNA) comprising the sequence of a preselected target sequence or a portion thereof, the method comprising contacting the target DNA with an engineered, non-naturally occurring system disclosed herein, thereby resulting in binding of the system to the target DNA.
  • a target nucleic acid e.g., DNA
  • This method can be useful, e.g., for detecting the presence and/or location of the a preselected target gene, for example, if a component of the system (e.g., the Cas protein) comprises a detectable marker.
  • a target nucleic acid e.g., DNA
  • a structure e.g., protein
  • the method comprising contacting the target DNA with an engineered, non-naturally occurring system disclosed herein, wherein the Cas protein comprises an effector domain or is associated with an effector protein, thereby resulting in modification of the target DNA or the structure associated with the target DNA.
  • the modification corresponds to the function of the effector domain or effector protein. Exemplary functions described in the “Cas Proteins” subsection in Section I supra are applicable hereto.
  • a method comprises contacting the target nucleic acid with a CRISPR-Cas complex comprising a targeter nucleic acid, a modulator nucleic acid, and a Cas protein disclosed herein.
  • the Cas protein is a type V-A, type V-C, or type V-D Cas protein (e.g., Cas nuclease).
  • the Cas protein is a type V-A Cas protein (e.g., Cas nuclease).
  • a method of editing a human genomic sequence at one of a group of preselected target gene loci comprising delivering an engineered, non-naturally occurring system disclosed herein into a human cell, thereby resulting in editing of the genomic sequence at the target gene locus in the human cell.
  • a method of detecting a human genomic sequence at one of a group of preselected target gene loci comprising delivering the engineered, non- naturally occurring system disclosed herein into a human cell, wherein a component of the system (e.g., the Cas protein) comprises a detectable marker, thereby detecting the target gene locus in the human cell.
  • a method of modifying a human chromosome at one of a group of preselected target gene loci comprising delivering the engineered, non-naturally occurring system disclosed herein into a human cell, wherein the Cas protein comprises an effector domain or is associated with an effector protein, thereby resulting in modification of the chromosome at the target gene locus in the human cell.
  • the CRISPR-Cas complex may be delivered to a cell by introducing a pre-formed ribonucleoprotein (RNP) complex into the cell. Alternatively, one or more components of the CRISPR-Cas complex may be expressed in the cell.
  • RNP ribonucleoprotein
  • Exemplary methods of delivery are known in the art and described in, for example, U.S. Patent Nos.8,697,359, 10,113,167, 10,570,418, 10,829,787, 11,118,194, and 11,125,739 and U.S. Patent Application Publication Nos. 2015/0344912, 2018/0119140, and 2018/0282763.
  • contacting a DNA e.g., genomic DNA
  • a CRISPR- Cas complex does not require delivery of all components of the complex into the cell.
  • one or more of the components may be pre-existing in the cell.
  • the cell (or a parental/ancestral cell thereof) has been engineered to express the Cas protein, and the single guide nucleic acid (or a nucleic acid comprising a regulatory element operably linked to a nucleotide sequence encoding the single guide nucleic acid), the targeter nucleic acid (or a nucleic acid comprising a regulatory element operably linked to a nucleotide sequence encoding the targeter nucleic acid), and/or the modulator nucleic acid (or a nucleic acid comprising a regulatory element operably linked to a nucleotide sequence encoding the modulator nucleic acid) are delivered into the cell.
  • the single guide nucleic acid or a nucleic acid comprising a regulatory element operably linked to a nucleotide sequence encoding the single guide nucleic acid
  • the targeter nucleic acid or a nucleic acid comprising a regulatory element operably linked to a nucleotide sequence encoding the targeter nucleic
  • the cell (or a parental/ancestral cell thereof) has been engineered to express the modulator nucleic acid, and the Cas protein (or a nucleic acid comprising a regulatory element operably linked to a nucleotide sequence encoding the Cas protein) and the targeter nucleic acid (or a nucleic acid comprising a regulatory element operably linked to a nucleotide sequence encoding the targeter nucleic acid) are delivered into the cell.
  • the Cas protein or a nucleic acid comprising a regulatory element operably linked to a nucleotide sequence encoding the Cas protein
  • the targeter nucleic acid or a nucleic acid comprising a regulatory element operably linked to a nucleotide sequence encoding the targeter nucleic acid
  • the cell (or a parental/ancestral cell thereof) has been engineered to express the Cas protein and the modulator nucleic acid, and the targeter nucleic acid (or a nucleic acid comprising a regulatory element operably linked to a nucleotide sequence encoding the targeter nucleic acid) is delivered into the cell.
  • the target DNA is in the genome of a target cell.
  • the present invention also provides a cell comprising the non-naturally occurring system or a CRISPR expression system described herein.
  • the present invention provides a cell whose genome has been modified by the CRISPR-Cas system or complex disclosed herein.
  • the target cells can be mitotic or post-mitotic cells from any organism, such as a bacterial cell (e.g., E coli), an archaeal cell, a cell of a single-cell eukaryotic organism, a plant cell, an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, or the like, a fungal cell (e.g., a yeast cell, such as S. cervisiae), an animal cell, a cell from an invertebrate animal (e.g.
  • a bacterial cell e.g., E coli
  • an archaeal cell e.g., a cell of a single-cell eukaryotic organism
  • a plant cell e.g., an algal cell, e.g., Botryococc
  • fruit fly enidarian, echinoderm, nematode, etc.
  • a cell from a vertebrate animal e.g., fish, amphibian, reptile, bird, mammal
  • a cell from a mammal e.g., a cell from a rodent, or a cell from a human.
  • target cells include but are not limited to a stem cell (e.g., an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a germ cell), a somatic cell (e.g., a fibroblast, a hematopoietic cell, a T lymphocyte (e.g., CD8+ T lymphocyte), an NK cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell), an in vitro or in vivo embryonic cell of an embryo at any stage (e.g., a 1-cell, 2-cell, 4-cell, 8-cell; stage zebrafish embryo).
  • a stem cell e.g., an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a germ cell
  • a somatic cell e.g., a fibroblast, a hematopoietic cell, a T lymphocyte (e.g., CD8
  • Cells may be from established cell lines or may be primary cells (i.e., cells and cells cultures that have been derived from a subject and allowed to grow in vitro for a limited number of passages of the culture).
  • primary cultures are cultures that may have been passaged within 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times to go through the crisis stage.
  • the primary cell lines are maintained for fewer than 10 passages in vitro. If the cells are primary cells, they may be harvest from an individual by any suitable method.
  • leukocytes may be harvested by apheresis, leukocytapheresis, or density gradient separation, while cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, or stomach can be harvested by biopsy.
  • the harvested cells may be used immediately, or may be stored under frozen conditions with a cryopreservative and thawed at a later time in a manner as commonly known in the art.
  • Ribonucleoprotein (RNP) delivery and “cas RNA” delivery An engineered, non-naturally occurring system disclosed herein can be delivered into a cell by suitable methods known in the art, including but not limited to ribonucleoprotein (RNP) delivery and “Cas RNA” delivery described below.
  • RNP ribonucleoprotein
  • Cas RNA delivery described below.
  • a CRISPR-Cas system including a single guide nucleic acid and a Cas protein, or a CRISPR-Cas system including a targeter nucleic acid, a modulator nucleic acid, and a Cas protein can be combined into a RNP complex and then delivered into the cell as a pre-formed complex.
  • a “ribonucleoprotein” or “RNP,” as used herein, can refer to a complex comprising a nucleoprotein and a ribonucleic acid.
  • nucleoprotein as provided herein can refer to a protein capable of binding a nucleic acid (e.g., RNA, DNA). Where the nucleoprotein binds a ribonucleic acid it can be referred to as “ribonucleoprotein.”
  • the interaction between the ribonucleoprotein and the ribonucleic acid may be direct, e.g., by covalent bond, or indirect, e.g., by non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g.
  • Non-limiting examples of delivery methods or vehicles include microinjection, biolistic particles, liposomes (see, e.g., U.S. Patent No.10,829,787) such as molecular trojan horses liposomes that delivers molecules across the blood brain barrier (see, Pardridge et al. (2010) Cold Spring Harb. Protoc., doi:10.1101/pdb.prot5407), immunoliposomes, virosomes, polycations, lipid:nucleic acid conjugates, electroporation, nanoparticles, nanowires (see, Shalek et al.
  • the CRISPR-Cas system is delivered into a cell in the form of (a) a single guide nucleic acid or a combination of a targeter nucleic acid and a modulator nucleic acid, and (b) a DNA comprising a regulatory element operably linked to a Cas coding sequence.
  • nucleic acid comprising a regulatory element operably linked to a nucleotide sequence encoding a guide nucleic acid disclosed herein.
  • the nucleic acid comprises a regulatory element operably linked to a nucleotide sequence encoding a single guide nucleic acid; this nucleic acid alone can constitute a CRISPR expression system.
  • the nucleic acid comprises a regulatory element operably linked to a nucleotide sequence encoding a targeter nucleic acid.
  • pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the ⁇ -actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1 ⁇ promoter.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • SV40 promoter the dihydrofolate reductase promoter
  • ⁇ -actin promoter the phosphoglycerol kinase (PGK) promoter
  • PGK phosphoglycerol kinase
  • the nucleotide sequence encoding the Cas protein is codon optimized for expression in a prokaryotic cell, e.g., E coli, eukaryotic host cell, e.g., a yeast cell (e.g., S. cerevisiae), a mammalian cell (e.g., a mouse cell, a rat cell, or a human cell), or a plant cell.
  • a prokaryotic cell e.g., E coli
  • eukaryotic host cell e.g., a yeast cell (e.g., S. cerevisiae)
  • a mammalian cell e.g., a mouse cell, a rat cell, or a human cell
  • Various species exhibit particular bias for certain codons of a particular amino acid.
  • C. Donor templates [0190] Cleavage of a target nucleotide sequence in the genome of a cell by a CRISPR-Cas system or complex can activate DNA damage pathways, which may rejoin the cleaved DNA fragments by NHEJ or HDR. HDR requires a repair template, either endogenous or exogenous, to transfer the sequence information from the repair template to the target.
  • an engineered, non-naturally occurring system or CRISPR expression system further comprises a donor template.
  • the term “donor template” can refer to a nucleic acid designed to serve as a repair template at or near the target nucleotide sequence upon introduction into a cell or organism.
  • the donor template is complementary to a polynucleotide comprising the target nucleotide sequence or a portion thereof.
  • a donor template may overlap with one or more nucleotides of a target nucleotide sequences (e.g. about or more than about 1, 5, 10, 15, 20, 25, 30, 35, 40, or more nucleotides).
  • the nucleotide sequence of the donor template is typically not identical to the genomic sequence that it replaces. Rather, the donor template may contain one or more substitutions, insertions, deletions, inversions or rearrangements with respect to the genomic sequence, so long as sufficient homology is present to support homology-directed repair.
  • the donor template comprises a non-homologous sequence flanked by two regions of homology (i.e., homology arms), such that homology-directed repair between the target DNA region and the two flanking sequences results in insertion of the non-homologous sequence at the target region.
  • the second homology arm is at least 50% (e.g., at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to a sequence 3’ to the target nucleotide sequence.
  • the nearest nucleotide of the donor template is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, or more nucleotides from the target nucleotide sequence.
  • the donor template further comprises an engineered sequence not homologous to the sequence to be repaired. Such engineered sequence can harbor a barcode and/or a sequence capable of hybridizing with a donor template-recruiting sequence disclosed herein.
  • the donor template further comprises one or more mutations relative to the genomic sequence, wherein the one or more mutations reduce or prevent cleavage, by the same CRISPR-Cas system, of the donor template or of a modified genomic sequence with at least a portion of the donor template sequence incorporated.
  • the PAM adjacent to the target nucleotide sequence and recognized by the Cas nuclease is mutated to a sequence not recognized by the same Cas nuclease.
  • the target nucleotide sequence e.g., the seed region
  • the one or more mutations are silent with respect to the reading frame of a protein-coding sequence encompassing the mutated sites.
  • the donor template can be provided to the cell as single-stranded DNA, single- stranded RNA, double-stranded DNA, or double-stranded RNA. It is understood that a CRISPR- Cas system, such as a system disclosed herein, may possess nuclease activity to cleave the target strand, the non-target strand, or both. When HDR of the target strand is desired, a donor template having a nucleic acid sequence complementary to the target strand is also contemplated. [0196] The donor template can be introduced into a cell in linear or circular form.
  • the ends of the donor template may be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art.
  • one or more dideoxynucleotide residues are added to the 3′ terminus of a linear molecule and/or self- complementary oligonucleotides are ligated to one or both ends (see, for example, Chang et al. (1987) PROC. NATL. ACAD SCI USA, 84: 4959; Nehls et al. (1996) SCIENCE, 272: 886; see also the chemical modifications for increasing stability and/or specificity of RNA disclosed supra).
  • Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues.
  • additional lengths of sequence may be included outside of the regions of homology that can be degraded without impacting recombination.
  • a donor template can be a component of a vector as described herein, contained in a separate vector, or provided as a separate polynucleotide, such as an oligonucleotide, linear polynucleotide, or synthetic polynucleotide.
  • the donor template is a DNA.
  • a donor template is in the same nucleic acid as a sequence encoding the single guide nucleic acid, a sequence encoding the targeter nucleic acid, a sequence encoding the modulator nucleic acid, and/or a sequence encoding the Cas protein, where applicable.
  • a donor template is provided in a separate nucleic acid.
  • a donor template polynucleotide may be of any suitable length, such as about or at least about 50, 75, 100, 150, 200, 500, 1000, 2000, 3000, 4000, or more nucleotides in length.
  • a donor template can be introduced into a cell as an isolated nucleic acid.
  • a donor template can be introduced into a cell as part of a vector (e.g., a plasmid) having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance, that are not intended for insertion into the DNA region of interest.
  • a donor template can be delivered by viruses (e.g., adenovirus, adeno-associated virus (AAV)).
  • viruses e.g., adenovirus, adeno-associated virus (AAV)
  • the donor template is introduced as an AAV, e.g., a pseudotyped AAV.
  • the capsid proteins of the AAV can be selected by a person skilled in the art based upon the tropism of the AAV and the target cell type.
  • the donor template is introduced into a hepatocyte as AAV8 or AAV9.
  • the donor template is introduced into a hematopoietic stem cell, a hematopoietic progenitor cell, or a T lymphocyte (e.g., CD8 + T lymphocyte) as AAV6 or an AAVHSC (see, U.S. Patent No.9,890,396).
  • sequence of a capsid protein may be modified from a wild-type AAV capsid protein, for example, having at least 50% (e.g., at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to a wild-type AAV capsid sequence.
  • the donor template can be delivered to a cell (e.g., a primary cell) by various delivery methods, such as a viral or non-viral method disclosed herein.
  • a non- viral donor template is introduced into the target cell as a naked nucleic acid or in complex with a liposome or poloxamer.
  • a non-viral donor template is introduced into the target cell by electroporation.
  • a viral donor template is introduced into the target cell by infection.
  • the engineered, non-naturally occurring system can be delivered before, after, or simultaneously with the donor template (see, International (PCT) Application Publication No. WO 2017/053729). A skilled person in the art will be able to choose proper timing based upon the form of delivery (consider, for example, the time needed for transcription and translation of RNA and protein components) and the half-life of the molecule(s) in the cell.
  • the donor template e.g., as an AAV
  • the donor template is introduced into the cell within 4 hours (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 90, 120, 150, 180, 210, or 240 minutes) after the introduction of the engineered, non-naturally occurring system.
  • the donor template is conjugated covalently to a modulator nucleic acid. Covalent linkages suitable for this conjugation are known in the art and are described, for example, in U.S.
  • the donor template is covalently linked to a modulator nucleic acid (e.g., the 5’ end of the modulator nucleic acid) through an internucleotide bond.
  • the donor template is covalently linked to a modulator nucleic acid (e.g., the 5’ end of the modulator nucleic acid) through a linker.
  • the donor template can comprise any nucleic acid chemistry.
  • the donor template can comprise DNA and/or RNA nucleotides.
  • the donor template can comprise single-stranded DNA, linear single- stranded RNA, linear double-stranded DNA, linear double-stranded RNA, circular single- stranded DNA, circular single-stranded RNA, circular double-stranded DNA, or circular double- stranded RNA.
  • the donor template comprises a mutation in a PAM sequence to partially or completely abolish binding of the RNP to the DNA.
  • the donor template is present at a concentration of at least 0.05, 0.01, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 3, or 4, and/or no more than 0.01, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 3, 4, or 5 ⁇ g ⁇ L -1 , for example 0.01-5 ⁇ g ⁇ L -1 .
  • the donor template comprises one or more promoters.
  • the donor template comprises a promoter that is at least 70, 75, 80, 85, 90, 95, 99.5, or 100% identical to any one of SEQ ID NOs: 78-85 of Table 6.
  • TABLE 6 Promoter sequences D.
  • Efficiency and specificity An engineered, non-naturally occurring system can be evaluated in terms of efficiency and/or specificity in nucleic acid targeting, cleavage, or modification. [0203] In certain embodiments, an engineered, non-naturally occurring system has high efficiency.
  • the genomes of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% of a population of cells, when the engineered, non-naturally occurring system is delivered into the cells, are targeted, cleaved, or modified.
  • the frequency of off-target events e.g., targeting, cleavage, or modification, depending on the function of the CRISPR-Cas system
  • off-target events were summarized in Lazzarotto et al. (2016) Nat Protoc.13(11): 2615-42, and include discovery of in situ Cas off-targets and verification by sequencing (DISCOVER-seq) as disclosed in Wienert et al.
  • transgenes due to the complex transcriptional regulation of genes in mammalian cells through networks of cis and trans regulatory elements, such as proximal and distal enhancers, and multiple transcription factors, attempts to alter the default genomic architecture by integration of exogenous DNA, e.g., transgenes, or synthetic sequences can affect the expression of the transgene itself leading to complete attenuation or complete silencing, and/or the expression of both nearby and distant endogenous genes that can, e.g., compromise the safety checkpoints that healthy cells have including dysregulation of expression of key genes, such as oncogenes and tumor suppressor genes, that can alter cellular behavior in dramatic ways, i.e., promoting clonal expansion or malignant transformation of the host.
  • exogenous DNA e.g., transgenes, or synthetic sequences
  • key genes such as oncogenes and tumor suppressor genes
  • Expression of exogenous genes, e.g., transgenes, in desired cell types and/or developmental/differentiation stages relies on integration into suitable target polynucleotide comprising a target nucleotide sequence that results in sufficient expression, to a degree sufficient for the intended purpose, from the candidate locus.
  • suitable target polynucleotide comprising a target nucleotide sequence that results in sufficient expression, to a degree sufficient for the intended purpose, from the candidate locus.
  • Expression from a specific genomic site can be affected by many factors including but not limited to cell type and differentiation stage, as one or more components of the target polynucleotide get activated during differentiation while others get silenced, and changes in chromatin architecture.
  • compositions and methods for genome engineering comprise compositions.
  • Certain embodiments comprise composition for editing genomes.
  • gNAs e.g., gRNAs
  • gRNAs that are complementary to a target nucleotide sequence in a target polynucleotide into which insertion of exogenous DNA, e.g., a transgene, doesn’t negatively affect the cell, e.g., significantly affect the expression of one or more endogenous genes or result in a malignant transformation of the cell.
  • gene expression demonstrated in the human target cell is maintained through differentiation of the human target cell and/or through proliferation in the one or more progeny cells at a level sufficient for the ultimate use of the cells.
  • Certain embodiments disclosed herein concern novel nucleic acid-guided nuclease complexes, e.g., RNPs, such as Cas bound to a gNA, that are complementary to a target nucleotide sequence within a target polynucleotide and hydrolyze the phosphodiester back bone (also referred as cleave or cut) in at least one position on at least one strand of the target polynucleotide.
  • Certain embodiments disclosed herein concern methods for selecting and using gNAs, e.g., gRNAs, for genome engineering.
  • Certain embodiments concern methods for using gNAs that are complementary to a target nucleotide sequence within a target polynucleotide, synthesizing the gNA and nucleic-acid-guided nuclease, and/or combining the nucleic guided nuclease with the gNA to form a nucleic acid-guided nuclease complex, e.g., RNP.
  • Certain embodiments disclosed herein concern methods.
  • Certain embodiments disclosed herein concern methods for engineering genomes.
  • nucleic acid-guided nuclease complex e.g., RNP
  • a donor template e.g., an exogenous DNA, e.g., a transgene
  • the nucleic-acid guided nuclease cleaves the backbone at a least one position in at least one of the strands of the target polynucleotide and the donor template is used to repair the cleaved target polynucleotide, introducing at least a portion of the donor template into the target polynucleotide.
  • exogenous DNA or a “transgene” includes any gene, natural or synthetic, which is introduced into the genome of an organism or cell to which it is not endogenous.
  • the transgene may or may not retain the ability to be expressed and/or produce RNA or protein in the human target cell.
  • the transgene may or may not alter the resulting phenotype of the human target cell.
  • a “human target cell” includes a cell into which an exogenous product, e.g., a protein, a nucleic acid, or a combination thereof, has been introduced.
  • a human target cell may be used to produce a gene product from an exogenous DNA, e.g., a transgene, such as an exogenous protein, e.g., a CAR.
  • a human target cell may comprise a target nucleotide sequence within target polynucleotide wherein a nucleic acid-guided nuclease hybridizes and cleaves at a site of cleavage at one or more positions on one or more strands of the target polynucleotide at or near the target nucleotide sequence.
  • a “site of cleavage” includes the location or locations at which a nucleic acid-guided nuclease complex will hydrolyze the phosphodiester backbone of a single- stranded or double-stranded target polynucleotide, after binding at a target nucleotide sequence in the target polynucleotide.
  • binding of the nucleic acid-guided nuclease complex to a target nucleotide sequence within the target polynucleotide can result in hydrolysis of one of the strands of the target polynucleotide at or near the target nucleotide sequence, resulting in strand cleavage.
  • the nucleic acid-guided nuclease complex can cleave either strand of the target polynucleotide.
  • binding of the nucleic acid-guided nuclease complex to a target nucleotide sequence within a target polynucleotide can result in hydrolysis of both strands of the target polynucleotide at or near the target nucleotide sequence, resulting in cleavage of both strands.
  • the sites of cleavage can be the same for both strands, resulting in a blunt end, or the sites of cleavage for each strand can be offset resulting in single strand overhangs, e.g., sticky ends.
  • mismatches at or near the site of cleavage may or may not affect the cleavage efficiency of the nucleic acid-guided nuclease complex.
  • Exemplary characteristics of a target nucleotide sequence that can demonstrate predictable function without potentially harmful alterations in human target cell genomic activity include one or more of (1) >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene, (2) >150 kb, for example, >200, such as >250, and in some cases >300 kb away from any miRNA/other functional small RNA, (3) >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end, (4) >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any replication origin, (5) >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any ultra-conserved element, (6) demonstrating low transcriptional activity, (7) outside of a copy number variable region, (8) located in open chromatin, and (9) unique
  • compositions In certain embodiments, provided herein are compositions. In certain embodiments, provided herein are compositions for engineering a human target cell at suitable target nucleotide sequences within a target polynucleotide of the human target cell. [0224] In certain embodiments, a suitable target polynucleotide that comprises a target nucleotide sequence has at least one of the exemplary characteristics. In certain embodiments, a suitable target polynucleotide that comprises a target nucleotide sequence has at least two of the exemplary characteristics. In certain embodiments, a suitable target polynucleotide that comprises a target nucleotide sequence has at least three of the exemplary characteristics.
  • a suitable target polynucleotide that comprises a target nucleotide sequence has at least four of the exemplary characteristics. In certain embodiments, a suitable target polynucleotide that comprises a target nucleotide sequence has at least five of the exemplary characteristics. In certain embodiments, a suitable target polynucleotide that comprises a target nucleotide sequence has at least six of the exemplary characteristics. In certain embodiments, a suitable target polynucleotide that comprises a target nucleotide sequence has at least seven of the exemplary characteristics. In certain embodiments, a suitable target polynucleotide that comprises a target nucleotide sequence has at least eight of the exemplary characteristics.
  • a suitable target polynucleotide that comprises a target nucleotide sequence has all the exemplary characteristics.
  • a suitable target polynucleotide is >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end.
  • a suitable target polynucleotide is >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end and further comprises at one additional exemplary characteristic.
  • a suitable target polynucleotide is >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end and further comprises at least two additional exemplary characteristics.
  • a suitable target polynucleotide is >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end and further comprises at least three additional exemplary characteristics.
  • a suitable target polynucleotide is >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end and further comprises at least four additional exemplary characteristics.
  • a suitable target polynucleotide is >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end and further comprises at least five additional exemplary characteristics.
  • a suitable target polynucleotide is >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end and further comprises at least six additional exemplary characteristics.
  • a suitable target polynucleotide is >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end and further comprises at least seven additional exemplary characteristics.
  • a suitable target polynucleotide is >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end and further comprises all eight additional exemplary characteristics.
  • a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene.
  • a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene and further comprises at one additional exemplary characteristic.
  • a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene and further comprises at least two additional exemplary characteristics.
  • a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene and further comprises at least three additional exemplary characteristics.
  • a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene and further comprises at least four additional exemplary characteristics.
  • a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene and further comprises at least five additional exemplary characteristics.
  • a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene and further comprises at least six additional exemplary characteristics.
  • a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene and further comprises at least seven additional exemplary characteristics.
  • a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene and further comprises all eight additional exemplary characteristics.
  • a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene, and >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end.
  • a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene, >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end, and further comprises at least one additional exemplary characteristic.
  • a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene, >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end, and further comprises at least two additional exemplary characteristics.
  • a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene, >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end, and further comprises at least three additional exemplary characteristics.
  • a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene, >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end, and further comprises at least four additional exemplary characteristics.
  • a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene, >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end, and further comprises at least five additional exemplary characteristics.
  • a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene, >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end, and further comprises at least six additional exemplary characteristics.
  • a suitable target polynucleotide is >150 kb, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene, >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end, and further comprises all seven additional exemplary characteristics.
  • a suitable target polynucleotide is >10 kb, for example, >20, such as >30, and in some cases >50 kb away from any 5’ gene end and >150, for example, >200, such as >250, and in some cases >300 kb away from a known cancer-related gene.
  • a suitable target polynucleotide comprising a target nucleotide sequence may comprise any one of SEQ ID NOs: 2020- 2043 of Table 7.
  • a suitable target polynucleotide comprising a target nucleotide sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or completely identical to any one of SEQ ID NOs: 2020-2043.
  • a suitable target polynucleotide comprising a target nucleotide sequence is at least 98% identical to any one of SEQ ID NOs: 2020-2043. In a more preferred embodiment, a suitable target polynucleotide comprising a target nucleotide sequence is at least 99% identical to any one of SEQ ID NOs: 2020-2043. [0230] In certain embodiments, a suitable target polynucleotide comprising a target nucleotide sequence, e.g., for transgene insertion, may comprise any one of SEQ ID NOs: 2020- 2042 of Table 7.
  • a suitable target polynucleotide comprising a target nucleotide sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or completely identical to any one of SEQ ID NOs: 2020-2042.
  • a suitable target polynucleotide comprising a target nucleotide sequence is at least 98% identical to any one of SEQ ID NOs: 2020-2042. In a more preferred embodiment, a suitable target polynucleotide comprising a target nucleotide sequence is at least 99% identical to any one of SEQ ID NOs: 2020-2042. [0231] In certain embodiments, a suitable target polynucleotide comprising a target nucleotide sequence, e.g., for transgene insertion, may comprise any one of SEQ ID NOs: 2020- 2041 and 2043 of Table 7.
  • a suitable target polynucleotide comprising a target nucleotide sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or completely identical to any one of SEQ ID NOs: 2020-2041 and 2043.
  • a suitable target polynucleotide comprising a target nucleotide sequence is at least 98% identical to any one of SEQ ID NOs: 2020-2041 and 2043. In a more preferred embodiment, a suitable target polynucleotide comprising a target nucleotide sequence is at least 99% identical to any one of SEQ ID NOs: 2020-2041 and 2043. [0232] In certain embodiments, a suitable target polynucleotide comprising a target nucleotide sequence, e.g., for transgene insertion, may comprise any one of SEQ ID NOs: 2020- 2041 of Table 7.
  • a suitable target polynucleotide comprising a target nucleotide sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or completely identical to any one of SEQ ID NOs: 2020-2041.
  • a suitable target polynucleotide comprising a target nucleotide sequence is at least 98% identical to any one of SEQ ID NOs: 2020-2041. In a more preferred embodiment, a suitable target polynucleotide comprising a target nucleotide sequence is at least 99% identical to any one of SEQ ID NOs: 2020-2041.
  • a suitable target polynucleotide comprising a target nucleotide sequence may comprise at least a portion of, for example, nucleotides 1-495, 1-490, 1-485, 1-480, 1-475, 1-470, 1-465, 1-460, 1-455, 1-450, 1- 445, 1-440, 1-435, 1-430, 1-425, 1-420, 1-415, 1-410, 1-405, or 1-400, of any one of SEQ ID NOs: 2020-2030 of Table 7.
  • a suitable target polynucleotide comprising a target nucleotide sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or completely identical to the portion of any one of SEQ ID NOs: 2020- 2030.
  • a suitable target polynucleotide comprising a target nucleotide sequence may comprise at least a portion of, for example, nucleotides 5-500, 10-500, 15-500, 20-500, 25-500, 30-500, 35-500, 40-500, 45-500, 50-500, 55-500, 60-500, 65-500, 70-500, 75-500, 80-500, 85-500, 90-500, 95-500, or 100-500, of any one of SEQ ID NOs: 2031-2041 of Table 7.
  • a suitable target polynucleotide comprising a target nucleotide sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or completely identical to the portion of any one of SEQ ID NOs: 2031-2041.
  • suitable target polynucleotides comprising a target nucleotide sequence for transgene insertion
  • expression of an exogenous DNA, e.g., transgene, inserted in a target polynucleotide at or near a target nucleotide sequence may depend on cell type and differentiation stage, as one or more components of a target polynucleotide get activated during differentiation while others get silenced, which may or may not be correlated with rearrangements of the chromatin architecture reorganization during differentiation.
  • a suitable target polynucleotide comprising a target nucleotide sequence demonstrates suitable expression of an inserted exogenous DNA, e.g., transgene, throughout differentiation and clonal expansion.
  • Pharmaceutical compositions [0236] Provided herein is a composition (e.g., pharmaceutical composition) comprising a guide nucleic acid, an engineered, non-naturally occurring system, or a eukaryotic cell, such as a guide nucleic acid, an engineered, non-naturally occurring system, or a eukaryotic cell, disclosed herein.
  • the composition comprises an RNP comprising a guide nucleic acid, such as a guide nucleic acid disclosed herein, and a Cas protein (e.g., Cas nuclease).
  • the composition comprises a single guide nucleic acid, such as a single guide nucleic acid disclosed herein.
  • the composition comprises an RNP comprising the single guide nucleic acid, and a Cas protein (e.g., Cas nuclease).
  • the composition comprises an RNP comprising the targeter nucleic acid, the modulator nucleic acid, and a Cas protein (e.g., Cas nuclease).
  • the composition comprises a complex of a targeter nucleic acid and a modulator nucleic acid, such as a complex of a targeter nucleic acid and a modulator nucleic acid disclosed herein.
  • the composition comprises an RNP comprising the targeter nucleic acid, the modulator nucleic acid, and a Cas protein (e.g., Cas nuclease).
  • a method of producing a composition comprising incubating a single guide nucleic acid, such as a single guide nucleic acid disclosed herein, with a Cas protein, thereby producing a complex of the single guide nucleic acid and the Cas protein (e.g., an RNP).
  • the method further comprises purifying the complex (e.g., the RNP).
  • a method of producing a composition comprising incubating a targeter nucleic acid and a modulator nucleic acid, such as a targeter nucleic acid and a modulator nucleic acid disclosed herein, under suitable conditions, thereby producing a composition (e.g., pharmaceutical composition) comprising a complex of the targeter nucleic acid and the modulator nucleic acid.
  • a modulator nucleic acid such as a targeter nucleic acid and a modulator nucleic acid disclosed herein
  • the method further comprises incubating the targeter nucleic acid and the modulator nucleic acid with a Cas protein (e.g., the Cas nuclease that the targeter nucleic acid and the modulator nucleic acid are capable of activating or a related Cas protein), thereby producing a complex of the targeter nucleic acid, the modulator nucleic acid, and the Cas protein (e.g., an RNP).
  • a Cas protein e.g., the Cas nuclease that the targeter nucleic acid and the modulator nucleic acid are capable of activating or a related Cas protein
  • the method further comprises purifying the complex (e.g., the RNP).
  • a guide nucleic acid, an engineered, non-naturally occurring system, a CRISPR expression system, or a cell comprising such system or modified by such system disclosed herein is combined with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable can refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit-to-risk ratio.
  • compositions include buffers, carriers, and excipients suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, or the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.
  • the outer surface of the nanoparticle can be conjugated with a positively charged polymer (e.g., polyethylenimine, polylysine, polyserine) which allows for attachment (e.g., conjugation or entrapment) of payload.
  • a positively charged polymer e.g., polyethylenimine, polylysine, polyserine
  • the pharmaceutical composition comprises an organic nanoparticle (e.g., entrapment of the payload inside the nanoparticle).
  • Exemplary organic nanoparticles include, e.g., SNALP liposomes that contain cationic lipids together with neutral helper lipids which are coated with polyethylene glycol (PEG) and protamine and nucleic acid complex coated with lipid coating.
  • the pharmaceutical composition comprises a liposome, for example, a liposome disclosed in International (PCT) Application Publication No. WO 2015/148863.
  • the pharmaceutical composition comprises a targeting moiety to increase target cell binding or update of nanoparticles and liposomes.
  • targeting moieties include cell specific antigens, monoclonal antibodies, single chain antibodies, aptamers, polymers, sugars, and cell penetrating peptides.
  • the pharmaceutical composition comprises a fusogenic or endosome-destabilizing peptide or polymer.
  • a pharmaceutical composition may contain a sustained- or controlled-delivery formulation.
  • sustained- or controlled-delivery means such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art.
  • Sustained-release preparations may include, e.g., porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules.
  • Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly (2- hydroxyethyl-inethacrylate), ethylene vinyl acetate, or poly-D( ⁇ )-3-hydroxybutyric acid.
  • Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art.
  • a pharmaceutical composition of the invention can be administered by a variety of methods known in the art. The route and/or mode of administration vary depending upon the desired results. Administration can be intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered proximal to the site of the target.
  • the pharmaceutically acceptable carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • the active compound e.g., the guide nucleic acid, engineered, non-naturally occurring system, or CRISPR expression system disclosed herein
  • the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol), and suitable mixtures thereof.
  • Pharmaceutical formulations preferably are sterile. Sterilization can be accomplished by any suitable method, e.g., filtration through sterile filtration membranes.
  • compositions of the invention typically employ a therapeutically effective dose or efficacious dose of the guide nucleic acid, engineered, non- naturally occurring system, or CRISPR expression system disclosed herein is employed in the pharmaceutical compositions of the invention.
  • the compositions disclosed herein are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of the invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular compositions disclosed herein employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors.
  • Guide nucleic acids, engineered, non-naturally occurring systems, and the CRISPR expression systems, e.g., as disclosed herein, are useful for targeting, editing, and/or modifying the genomic DNA in a cell or organism.
  • guide nucleic acids and systems can be used to treat a disease or disorder in which modification of genetic or epigenetic information is desirable.
  • a method of treating a disease or disorder comprising administering to a subject in need thereof a guide nucleic acid, a non-naturally occurring system, a CRISPR expression system, or a cell disclosed herein.
  • subject includes human and non-human animals.
  • Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, and reptiles.
  • treatment can refer to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease or delaying the disease progression.
  • Treatment covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) inhibiting the disease, i.e., arresting its development; and (b) relieving the disease, i.e., causing regression of the disease.
  • the guide nucleic acid, the engineered, non-naturally occurring system, and the CRISPR expression system disclosed herein can be used to treat any suitable disease or disorder that can be improved by the system in a cell.
  • certain methods disclosed herein is particularly suitable for editing or modifying a proliferating cell, such as a stem cell (e.g., a hematopoietic stem cell), a progenitor cell (e.g., a hematopoietic progenitor cell or a lymphoid progenitor cell), or a memory cell (e.g., a memory T cell).
  • a stem cell e.g., a hematopoietic stem cell
  • a progenitor cell e.g., a hematopoietic progenitor cell or a lymphoid progenitor cell
  • a memory cell e.g., a memory T cell
  • the engineered, non-naturally occurring system of the present invention has the advantage of increasing or decreasing the efficiency of nucleic acid cleavage by, for example, adjusting the hybridization of dual guide nucleic acids. As a result, it can be used to minimize off-target events when creating genetically engineered proliferating cells.
  • the guide nucleic acid, the engineered, non-naturally occurring system, and/or the CRISPR expression system disclosed herein can be used to engineer an immune cell.
  • Immune cells include but are not limited to lymphocytes (e.g., B lymphocytes or B cells, T lymphocytes or T cells, and natural killer cells), myeloid cells (e.g., monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes), and the stem and progenitor cells that can differentiate into these cell types (e.g., hematopoietic stem cells, hematopoietic progenitor cells, and lymphoid progenitor cells).
  • lymphocytes e.g., B lymphocytes or B cells, T lymphocytes or T cells, and natural killer cells
  • myeloid cells e.g., monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes
  • the cells can include autologous cells derived from a subject to be treated, or alternatively allogenic cells derived from a donor.
  • the immune cell is a T cell, which can be, for example, a cultured T cell, a primary T cell, a T cell from a cultured T cell line (e.g., Jurkat, SupTi), or a T cell obtained from a mammal, for example, from a subject to be treated. If obtained from a mammal, the T cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T cells can also be enriched or purified.
  • the T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4 + /CD8 + double positive T cells, CD4 + helper T cells (e.g., Th1 and Th2 cells), CD8 + T cells (e.g., cytotoxic T cells), tumor infiltrating lymphocytes (TILs), memory T cells (e.g., central memory T cells and effector memory T cells), regulatory T cells, naive T cells, or the like.
  • an immune cell e.g., a T cell, is engineered to express an exogenous gene.
  • an engineered CRISPR system disclosed herein may catalyze DNA cleavage at the gene locus, allowing for site-specific integration of the exogenous gene at the gene locus by HDR.
  • an immune cell e.g., a T cell
  • a chimeric antigen receptor i.e., the T cell comprises an exogenous nucleotide sequence encoding a CAR.
  • the term “chimeric antigen receptor” or “CAR” includes any artificial receptor including an antigen-specific binding moiety and one or more signaling chains derived from an immune receptor.
  • CARs can comprise a single chain fragment variable (scFv) of an antibody specific for an antigen coupled via hinge and transmembrane regions to cytoplasmic domains of T cell signaling molecules, e.g. a T cell costimulatory domain (e.g., from CD28, CD137, OX40, ICOS, or CD27) in tandem with a T cell triggering domain (e.g. from CD3 ⁇ ).
  • T cell costimulatory domain e.g., from CD28, CD137, OX40, ICOS, or CD27
  • T cell triggering domain e.g. from CD3 ⁇
  • a T cell expressing a chimeric antigen receptor is referred to as a CAR T cell.
  • Exemplary CAR T cells include CD19 targeted CTL019 cells (see, Grupp et al. (2015) BLOOD, 126: 4983), 19-28z cells (see, Park et al. (2015) J. CLIN.
  • an immune cell e.g., a T cell
  • binds an antigen e.g., a cancer antigen
  • an endogenous T cell receptor TCR
  • an immune cell e.g., a T cell
  • is engineered to express an exogenous TCR e.g., an exogenous naturally occurring TCR or an exogenous engineered TCR.
  • T cell receptors comprise two chains referred to as the ⁇ - and ⁇ -chains, that combine on the surface of a T cell to form a heterodimeric receptor that can recognize MHC-restricted antigens.
  • Each of ⁇ - and ⁇ -chain comprises a constant region and a variable region.
  • Each variable region of the ⁇ - and ⁇ -chains defines three loops, referred to as complementary determining regions (CDRs) known as CDR 1 , CDR 2 , and CDR 3 that confer the T cell receptor with antigen binding activity and binding specificity.
  • CDRs complementary determining regions
  • a CAR or TCR binds a cancer antigen selected from B-cell maturation antigen (BCMA), mesothelin, prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD70, CD74, CD123, CD133, CD138, epithelial glycoprotein2 (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor-type tyrosine- protein kinase (FLT3), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a and ⁇ (FRa and ⁇ ), Ganglioside G2 (GD2),
  • BCMA B-cell matur
  • TCR subunit loci e.g., the TCR ⁇ constant (TRAC) locus, the TCR ⁇ constant 1 (TRBC1) locus, and the TCR ⁇ constant 2 (TRBC2) locus. It is understood that insertion in the TRAC locus reduces tonic CAR signaling and enhances T cell potency (see, Eyquem et al. (2017) NATURE, 543: 113).
  • an immune cell e.g., a T cell
  • an immune cell is engineered to have reduced expression of an endogenous TCR or TCR subunit, e.g., TRAC, TRBC1, and/or TRBC2.
  • the cell may be engineered to have partially reduced or no expression of the endogenous TCR or TCR subunit.
  • the immune cell e.g., a T cell
  • the immune cell is engineered to have less than 80% (e.g., less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%) of the expression of the endogenous TCR or TCR subunit relative to a corresponding unmodified or parental cell.
  • the immune cell e.g., a T cell
  • the immune cell is engineered to have no detectable expression of the endogenous TCR or TCR subunit. Exemplary approaches to reduce expression of TCRs using CRISPR systems are described in U.S. Patent No.9,181,527, Liu et al.
  • T cells also express major histocompatibility complex (MHC) or human leukocyte antigen (HLA) genes, and inactivation of these endogenous gene may reduce an immune response, thereby allowing use of allogeneic T cells as starting materials for preparation of CAR T cells.
  • MHC major histocompatibility complex
  • HLA human leukocyte antigen
  • an immune cell e.g., a T-cell
  • a T-cell is engineered to have reduced expression of one or more endogenous class I or class II MHCs or HLAs (e.g., beta 2-microglobulin (B2M), class II major histocompatibility complex transactivator (CIITA)).
  • the cell may be engineered to have partially reduced or no expression of an endogenous MHC or HLA.
  • the immune cell e.g., a T-cell
  • the immune cell is engineered to have less than less than 80% (e.g., less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%) of the expression of endogenous MHC (e.g., B2M, CIITA) relative to a corresponding unmodified or parental cell.
  • the immune cell e.g., a T cell
  • a cell may be engineered to have expression of, e.g., HLA-E and/or HLA-G, in order to avoid attack by natural killer (NK) cells.
  • HLA-E and/or HLA-G in order to avoid attack by natural killer (NK) cells.
  • NK natural killer
  • Exemplary approaches to reduce expression of MHCs using CRISPR systems are described in Liu et al. (2017) CELL RES, 27: 154, Ren et al. (2017) CLIN CANCER RES, 23: 2255, and Ren et al. (2017) ONCOTARGET, 8: 17002.
  • Other genes that may be inactivated include but are not limited to CD3, CD52, and deoxycytidine kinase (DCK).
  • inactivation of DCK may render the immune cells (e.g., T cells) resistant to purine nucleotide analogue (PNA) compounds, which are often used to compromise the host immune system in order to reduce a GVHD response during an immune cell therapy.
  • the immune cell e.g., a T-cell
  • the immune cell is engineered to have less than less than 80% (e.g., less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%) of the expression of endogenous CD52 or DCK relative to a corresponding unmodified or parental cell.
  • an immune cell e.g., T cell
  • an immune cell is engineered to have reduced expression of an immune checkpoint protein.
  • immune checkpoint proteins expressed by wild-type T cells include but are not limited to PDCD1 (PD-1), CTLA4, ADORA2A (A2AR), B7-H3, B7-H4, BTLA, KIR, LAG3, HAVCR2 (TIM3), TIGIT, VISTA, PTPN6 (SHP-1), and FAS.
  • the cell may be modified to have partially reduced or no expression of the immune checkpoint protein.
  • the immune cell e.g., a T cell
  • the immune cell is engineered to have less than 80% (e.g., less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%) of the expression of the immune checkpoint protein relative to a corresponding unmodified or parental cell.
  • the immune cell e.g., a T cell
  • Exemplary approaches to reduce expression of immune checkpoint proteins using CRISPR systems are described in International (PCT) Publication No. WO 2017/017184, Cooper et al.
  • the immune cell can be engineered to have reduced expression of an endogenous gene, e.g., an endogenous genes described above, by gene editing or modification.
  • an engineered CRISPR system disclosed herein may result in DNA cleavage at a gene locus, thereby inactivating the targeted gene.
  • an engineered CRISPR system disclosed herein may be fused to an effector domain (e.g., a transcriptional repressor or histone methylase) to reduce the expression of the target gene.
  • the immune cell can also be engineered to express an exogenous protein (besides an antigen-binding protein described above) at the locus of a human ADORA2A, B2M, CD52, CIITA, CTLA4, DCK, FAS, HAVCR2, LAG3, PDCD1, PTPN6, TIGIT, TRAC, TRBC1, TRBC2, CARD11, CD247, IL7R, LCK, or PLCG1 gene.
  • an immune cell e.g., a T cell, is modified to express a dominant-negative form of an immune checkpoint protein.
  • the dominant-negative form of the checkpoint inhibitor can act as a decoy receptor to bind or otherwise sequester the natural ligand that would otherwise bind and activate the wild-type immune checkpoint protein.
  • engineered immune cells for example, T cells containing dominant-negative forms of an immune suppressor are described, for example, in International (PCT) Publication No. WO 2017/040945.
  • an immune cell e.g., a T cell
  • a gene e.g., a transcription factor, a cytokine, or an enzyme
  • a gene e.g., a transcription factor, a cytokine, or an enzyme
  • the immune cell is modified to express TET2, FOXO1, IL-12, IL-15, IL-18, IL-21, IL-7, GLUT1, GLUT3, HK1, HK2, GAPDH, LDHA, PDK1, PKM2, PFKFB3, PGK1, ENO1, GYS1, and/or ALDOA.
  • the modification is an insertion of a nucleotide sequence encoding the protein operably linked to a regulatory element.
  • the modification is a substitution of a single nucleotide polymorphism (SNP) site in the endogenous gene.
  • SNP single nucleotide polymorphism
  • Exemplary genetic diseases or disorders include age-related macular degeneration, adrenoleukodystrophy (ALD), Alagille syndrome, alpha-1-antitrypsin deficiency, argininemia, argininosuccinic aciduria, ataxia (e.g., Friedreich ataxia, spinocerebellar ataxias, ataxia telangiectasia, essential tremor, spastic paraplegia), autism, biliary atresia, biotinidase deficiency, carbamoyl phosphate synthetase I deficiency, carbohydrate deficient glycoprotein syndrome (CDGS), a central nervous system (CNS)-related disorder (e.g., Alzheimer's disease, amyotrophic lateral sclerosis (ALS), canavan disease (CD), ischemia, multiple sclerosis (MS), neuropathic pain, Parkinson's disease), Bloom's syndrome, cancer, Charcot-Marie-T
  • kits are useful for identifying a donor template that introduces optimal genetic modification in a multiplex assay.
  • the CRISPR expression systems as disclosed herein are also suitable for use in a kit.
  • a kit further comprises one or more reagents and/or buffers for use in a process utilizing one or more of the elements described herein.
  • Reagents may be provided in any suitable container and may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g., in concentrate or lyophilized form).
  • Embodiments [0281] in embodiment 1 provided herein is a polypeptide that is at least 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5% identical, or 100% identical to any one of SEQ ID NOs: 86-124 or 2044-2070.
  • embodiment 2 provided herein is the polypeptide of embodiment 1, wherein the polypeptide is at least 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5% identical, or 100% identical to any one of SEQ ID NOs: 86-104, 116-124, or 2044-2070.
  • embodiment 3 provided herein is the polypeptide of any one of embodiments 1 or 2, wherein the sequence identity is at least 90%.
  • polypeptide of any one of embodiments 1 or 2 wherein the sequence identity is at least 95%.
  • sequence identity is at least 99%.
  • sequence identity is at least 99.5%.
  • sequence identity is 100%.
  • embodiment 8 provided herein is a polynucleotide encoding a polypeptide of any one of embodiments 1 through 7.
  • polynucleotide of embodiment 10 wherein the polynucleotide further comprises a second portion comprising a second sequence encoding a second polypeptide comprising a second CAR or portion thereof different from the first CAR or portion thereof.
  • first and second polypeptides are separate polypeptides.
  • embodiment 13 provided herein is the polynucleotide of embodiment 11, wherein the first and second polypeptides are linked.
  • embodiment 14 provided herein is the polynucleotide of embodiment 13, wherein the first and second polypeptides are linked by one or more amino acids.
  • polynucleotide of embodiment 14 wherein the second CAR or portion thereof binds to a different site on the same binding partner.
  • embodiment 19 provided herein is the polynucleotide of embodiment 18, wherein the first or second polypeptide comprise a polypeptide that is at least 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5% identical, or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 86-104, 116-124, or 2044-2070.
  • a cell comprising a first polynucleotide of any one of embodiments 10 through 19.
  • the cell further comprises a second polynucleotide of any one of embodiments 10 through 19, wherein the second polynucleotide is different from the first.
  • embodiment 22 provided herein is the cell of any one of embodiments 20 or 21, wherein the first and/or second polynucleotides further comprise homology arms flanking the first and/or second sequences encoding the first and/or second polypeptides.
  • embodiment 23 provided herein is the cell of any one of embodiments 20 or 22, further comprising a nucleic acid-guided nuclease.
  • embodiment 24 provided herein is the cell of embodiment 23, wherein the nucleic acid-guided nuclease comprises an engineered, non-naturally occurring nuclease.
  • embodiment 25 provided herein is the cell of any one of embodiments 23 or 24, wherein the nucleic acid-guided nuclease comprises a Class 1 or a Class 2 nuclease.
  • nucleic acid-guided nuclease comprises a Type II or Type V nuclease.
  • nucleic acid-guided nuclease comprises a Type V-A, V-B, V-C, V-D, or V-E nuclease.
  • nucleic acid-guided nuclease comprises a Type V-A nuclease.
  • nucleic acid-guided nuclease comprises a MAD nuclease, an ART nuclease, or an ABW nuclease.
  • nucleic acid-guided nuclease comprises an amino acid sequence at least 80, 85, 90, 95, 99, or 100% identical to an amino acid sequence of a MAD, ART, or ABW nuclease.
  • nucleic acid-guided nuclease comprises a MAD1, MAD2, MAD3, MAD4, MAD5, MAD6, MAD7, MAD8, MAD9, MAD10, MAD11, MAD12, MAD13, MAD14, MAD15, MAD16, MAD17, MAD18, MAD19, or MAD20 nuclease.
  • nucleic acid-guided nuclease comprises an ART1, ART2, ART3, ART4, ART5, ART6, ART7, ART8, ART9, ART10, ART11, ART11*, ART12, ART13, ART14, ART15, ART16, ART17, ART18, ART19, ART20, ART21, ART22, ART23, ART24, ART25, ART26, ART27, ART28, ART29, ART30, ART31, ART32, ART33, ART34, or ART35 nuclease.
  • nucleic acid-guided nuclease comprises an amino acid sequence at least 80, 85, 90, 95, 99% identical, or 100% identical to the amino acid sequence of MAD2, MAD7, ART2, ART11, or ART11*.
  • nucleic acid-guided nuclease comprises an amino acid sequence that is at least 80, 85, 90, 95, 99% identical, or 100% identical to the amino acid sequence of SEQ ID NO: 37.
  • nucleic acid-guided nuclease comprises five N-terminal NLS.
  • nucleic acid-guided nuclease comprises five N-terminal NLS.
  • embodiment 40 provided herein is the cell of any one of embodiments 35 through 39, wherein the NLS comprise any one of SEQ ID NOs: 40-56.
  • embodiment 41 provided herein is the cell of embodiment 40, wherein the NLS comprises any one of SEQ ID NOs: 40, 51, and 56.
  • embodiment 42 provided herein is the cell of any one of embodiments 20 through 41, further comprising a guide nucleic acid (gNA).
  • gNA guide nucleic acid
  • the gNA comprises: (i) a targeter nucleic acid comprising a targeter stem sequence and a spacer sequence; and (ii) a modulator nucleic acid comprising a modulator stem sequence complementary to the targeter stem sequence, and, optionally, a 5' sequence.
  • the gNA an engineered, non-naturally occurring guide nucleic acid.
  • the gNA comprises a single polynucleotide.
  • embodiment 46 is the cell of any one of embodiments 43 or 44, wherein the gNA comprises a dual guide nucleic acid, wherein the targeter nucleic acid and the modulator nucleic acid are separate polynucleotides.
  • embodiment 47 provided herein is the cell of embodiment 46, wherein the dual gNA is capable of binding to and activating a nucleic acid-guided nuclease, that, in a naturally occurring system, is activated by a single crRNA in the absence of a tracrRNA.
  • embodiment 48 provided herein is the cell of any one of embodiments 42 through 47, wherein the gNA and the nucleic acid-guided nuclease form a nucleic acid-guided nuclease complex.
  • embodiment 49 is the cell of one of embodiments 42 through 48, wherein the gNA further comprises a donor template recruiting sequence.
  • embodiment 50 provided herein is the cell of any one of embodiments 20 through 49, wherein the first and/or second polynucleotides are integrated into first and/or second locations in a genome of the cell.
  • embodiment 51 provided herein is the cell of embodiment 50, wherein the first and/or second location in the genome of the cell comprises a safe harbor site or a gene coding for a subunit of an HLA-1, HLA-2, or TRC protein.
  • embodiment 52 provided herein is the cell of embodiment 50, wherein the location of the genome of the cell comprises a TRAC gene.
  • embodiment 53 provided herein is the cell of any one of embodiments 50 through 52, wherein the spacer sequence is at least partially complementary to a target sequence within or near the first and/or second locations in the genome of the cell.
  • embodiment 54 provided herein is the cell of embodiment 53, wherein the spacer sequence is at least 50, 60, 70, 80, 90, 95, 99, 99.5% identical, or 100% identical to the target sequence within or near the first and/or second locations in the genome of the cell.
  • embodiment 55 provided herein is the cell of any one of embodiments 20 through 54, wherein the first and/or second polypeptides are expressed.
  • embodiment 56 provided herein is the cell of embodiment 55, wherein the first and second polypeptides are expressed on the surface of the cell.
  • embodiment 57 provided herein is the cell of embodiment 55, wherein expression of the second polypeptide is initiated by a signal indicating a change in the state of the cell.
  • embodiment 58 provided herein is the cell of embodiment 57, wherein binding of a binding partner to the first polypeptide initiates the signal indicating a change in the state of the cell.
  • embodiment 59 provided herein is the cell of embodiment 55, wherein the first polypeptide is expressed on a surface of the cell and the second polypeptide is secreted.
  • embodiment 60 provided herein is the cell of embodiment 59, wherein expression of the second polypeptide is initiated by a signal indicating a change in the state of the cell.
  • embodiment 61 provided herein is the cell of embodiment 60, wherein binding of a binding partner to the first polypeptide initiates the signal indicating a change in the state of the cell.
  • embodiment 62 provided herein is the cell of any one of embodiments 20 through 61, wherein the cell is a human cell.
  • embodiment 63 provided herein is the cell of embodiment 62, wherein the human cell is an immune cell or a stem cell.
  • embodiment 64 provided herein is the cell of embodiment 62, wherein the human cell is an immune cell comprising a neutrophil, eosinophil, basophil, mast cell, monocyte, macrophage, dendritic cell, natural killer cell, or a lymphocyte.
  • embodiment 65 is the cell of embodiment 62, wherein the human cell is a T cell.
  • embodiment 66 provided herein is the cell of embodiment 62, wherein the human cell is a stem cell that is a human pluripotent, multipotent stem cell, embryonic stem cell, induced pluripotent stem cell, hematopoietic stem cell, CD34+ cell.
  • embodiment 67 provided herein is a cell comprising: (a) a first polynucleotide comprising a first portion comprising a first sequence encoding a first polypeptide comprising a first CAR or portion thereof.
  • embodiment 68 provided herein is the cell of embodiment 67, further comprising: (b) a nucleic acid-guided nuclease system, any/or one or more polynucleotides encoding one or more parts of the system, wherein the system comprises: (i) a nucleic acid-guided nuclease; and (ii) a guide nucleic acid (gNA) compatible with the nucleic acid-guided nuclease.
  • the nucleic acid-guided nuclease comprises a Type V nucleic acid-guided nuclease.
  • embodiment 70 is the cell of any one of embodiments 67 through 69, wherein the first polynucleotide comprises a donor template.
  • embodiment 71 provided herein is the cell of any one of embodiments 67 through 70, wherein the first polynucleotide further comprises a second portion comprising a second sequence encoding a second polypeptide comprising a second CAR or portion thereof.
  • embodiment 72 provided herein is the cell of any one of embodiments 67 through 70, wherein the cell further comprises a second polynucleotide comprising a second sequence encoding a second polypeptide comprising a second CAR or portion thereof.
  • embodiment 73 provided herein is the cell of any one of embodiments 71 or 72, wherein the second CAR or portion thereof is different from the first CAR or portion thereof.
  • embodiment 74 provided herein is the cell of any one of embodiments 67 through 73, wherein at least a portion of the first and/or second polynucleotides are inserted into a location in a genome of the cell.
  • embodiment 75 provided herein is the cell of embodiment 74, wherein the first polynucleotide is inserted into a first location in the genome of the cell and the second polynucleotide is inserted into a second location in the genome of the cell, wherein the first location is different from the second location.
  • embodiment 76 provided herein is the cell of any one of embodiments 67 through 75, wherein the first CAR or portion thereof comprises a polypeptide that binds to a binding partner comprising B7H3, BCMA, GPRC5D, CD8, CD8a, CD19, CD20, CD22, CD28, 4-1BB, or CD3zeta or a portion thereof.
  • a binding partner comprising B7H3, BCMA, GPRC5D, CD8, CD8a, CD19, CD20, CD22, CD28, 4-1BB, or CD3zeta or a portion thereof.
  • embodiment 77 provided herein is the cell of embodiment 76, wherein the second CAR or portion thereof comprises a polypeptide that binds to a binding partner comprising B7H3, BCMA, GPRC5D, CD8, CD8a, CD19, CD20, CD22, CD28, 4-1BB, or CD3zeta or a portion thereof, different from the binding partner of the first CAR or portion thereof.
  • a binding partner comprising B7H3, BCMA, GPRC5D, CD8, CD8a, CD19, CD20, CD22, CD28, 4-1BB, or CD3zeta or a portion thereof, different from the binding partner of the first CAR or portion thereof.
  • embodiment 78 provided herein is the cell of any one of embodiments 76 or 77, wherein the first CAR or portion thereof comprises a polypeptide at least 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5% identical, or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 86-124 or 2044-2070.
  • embodiment 79 provided herein is the cell of any one of embodiments 67 through 78, wherein the first and/or second polypeptides are expressed.
  • embodiment 80 provided herein is the cell of any one of embodiments 67 through 79, wherein the nucleic acid-guided nuclease comprises an engineered, non-naturally occurring nuclease.
  • nucleic acid-guided nuclease comprises a Type V-A, V-B, V-C, V-D, or V-E nuclease.
  • nucleic acid-guided nuclease comprises a Type V-A nuclease.
  • nucleic acid-guided nuclease comprises a MAD nuclease, an ART nuclease, or an ABW nuclease.
  • nucleic acid-guided nuclease comprises an amino acid sequence at least 80, 85, 90, 95, 99% identical, or 100% identical to an amino acid sequence of a MAD, ART, or ABW nuclease.
  • nucleic acid-guided nuclease comprises a MAD1, MAD2, MAD3, MAD4, MAD5, MAD6, MAD7, MAD8, MAD9, MAD10, MAD11, MAD12, MAD13, MAD14, MAD15, MAD16, MAD17, MAD18, MAD19, or MAD20 nuclease.
  • nucleic acid-guided nuclease comprises an ART1, ART2, ART3, ART4, ART5, ART6, ART7, ART8, ART9, ART10, ART11, ART11*, ART12, ART13, ART14, ART15, ART16, ART17, ART18, ART19, ART20, ART21, ART22, ART23, ART24, ART25, ART26, ART27, ART28, ART29, ART30, ART31, ART32, ART33, ART34, or ART35 nuclease.
  • nucleic acid-guided nuclease comprises an amino acid sequence at least 80, 85, 90, 95, 99% identical, or 100% identical, to the amino acid sequence of MAD2, MAD7, ART2, ART11, or ART11*.
  • nucleic acid-guided nuclease comprises an amino acid sequence that is at least 80, 85, 90, 95, 99% identical, or 100% identical, to the amino acid sequence of SEQ ID NO: 37.
  • nucleic acid-guided nuclease further comprises a nuclear localization signal (NLS), a purification tag, and/or a cleavage site.
  • NLS nuclear localization signal
  • the nucleic acid-guided nuclease comprises at least four NLS.
  • the nucleic acid-guided nuclease comprises one N-terminal and three C-terminal NLS.
  • the nucleic acid-guided nuclease comprises at least five NLS.
  • nucleic-acid-guided nuclease comprises five N-terminal NLS.
  • nucleic-acid-guided nuclease comprises five N-terminal NLS.
  • NLS comprise any one of SEQ ID NOs: 40-56.
  • NLS comprises SEQ ID NOs: 40, 51, and 56.
  • embodiment 96 provided herein is the cell of any one of embodiments 67 through 95, wherein the gNA comprises: (1) provided herein is a targeter nucleic acid comprising a targeter stem sequence and a spacer sequence; and (2) provided herein is a modulator nucleic acid comprising a modulator stem sequence complementary to the targeter stem sequence, and, optionally, a 5' sequence.
  • embodiment 97 provided herein is the cell of any one of embodiments 67 through 96, wherein the gNA an engineered, non-naturally occurring guide nucleic acid.
  • embodiment 98 provided herein is the cell of any one of embodiments 67 through 97, wherein the gNA comprises a single polynucleotide.
  • embodiment 99 provided herein is the cell of any one of embodiments 67 through 97, wherein the gNA comprises a dual guide nucleic acid, wherein the targeter nucleic acid and the modulator nucleic acid are separate polynucleotides.
  • embodiment 100 provided herein is the cell of embodiment 99, wherein the dual gNA is capable of binding to and activating a nucleic acid-guided nuclease, that, in a naturally occurring system, is activated by a single crRNA in the absence of a tracrRNA.
  • embodiment 101 is the cell of any one of embodiments 67 through 100, wherein the gNA and the nucleic acid-guided nuclease form a nucleic acid-guided nuclease complex.
  • embodiment 102 provided herein is the cell of any one of embodiments 67 through 101, wherein the gNA further comprises a donor template recruiting sequence.
  • embodiment 103 provided herein is the cell of any one of embodiments 67 through 102, wherein the cell is a human cell.
  • embodiment 104 provided herein is the cell of embodiment 103, wherein the human cell is an immune cell or a stem cell.
  • embodiment 105 is the cell of embodiment 103, wherein the human cell is an immune cell comprising a neutrophil, eosinophil, basophil, mast cell, monocyte, macrophage, dendritic cell, natural killer cell, or a lymphocyte.
  • the human cell is a T cell.
  • the human cell is a stem cell that is a human pluripotent, multipotent stem cell, embryonic stem cell, induced pluripotent stem cell, hematopoietic stem cell, CD34+ cell.
  • embodiment 108 provided herein is the cell of embodiment 103, wherein the human cell is an induced pluripotent stem cell.
  • embodiment 109 provided herein is the cell of any one of embodiments 103 through 108, wherein the cell is a cell demonstrating reduced immunogenicity when placed in an allogeneic host.
  • embodiment 110 provided herein is the cell of embodiment 109, wherein the cell is non- immunogenic when placed in an allogeneic host.
  • compositions comprising a plurality of cell populations comprising a first and a second cell population wherein: (a) the first cell population comprises (i) a first genomic modification comprising insertion of a first polynucleotide encoding a first CAR or portion thereof at a first location of the genome; and (ii) a second genomic modification comprising insertion of a second polynucleotide encoding a second CAR or portion thereof at a second location of the genome; and (b) a second cell population comprises the first genomic modification or the second genomic modification but not both.
  • embodiment 112 provided herein is the composition of embodiment 111, further comprising a third cell population different from the first and the second cell populations, wherein the third cell population comprises the first genomic modification or the second genomic modification but not both.
  • embodiment 113 provided herein is the composition of embodiment 112, further comprising a fourth cell population different from the first, second, and third cell populations.
  • embodiment 114 provided herein is a composition comprising a pharmaceutically acceptable excipient and a composition of any one of embodiments 20 through 113.
  • composition 115 comprising: (a) a first Type V nucleic acid guided nuclease; (b) a first guide nucleic acid (gNA); and (c) a first donor template comprising a first polynucleotide encoding a first polypeptide comprising a first CAR or portion thereof.
  • a second Type V nucleic acid guided nuclease comprising a second gNA; and (f) a second donor template comprising a second polynucleotide encoding a second polypeptide comprising a CAR or portion thereof.
  • embodiment 117 provided herein is the composition of embodiment 116, further comprising: (g) a third Type V nucleic acid guided nuclease; (h) a third gNA; and (i) a third donor template comprising a third polynucleotide different from the first and or second polynucleotides.
  • embodiment 118 provided herein is the composition of any one of embodiments 115 through 117, wherein the nucleic acid guided-nuclease comprises MAD7.
  • embodiment 119 provided herein is the composition of any one of embodiments 115 through 118, wherein the gNA comprises a dual gNA.
  • embodiment 120 provided herein is the composition of any one of embodiments 115 through 119, wherein the first and second polypeptides comprise a CAR or portion thereof that binds a binding partner comprising B7H3, BCMA, GPRC5D, CD8, CD8a, CD19, CD20, CD22, CD28, 4-1BB, or CD3zeta or a portion thereof.
  • embodiment 121 provided herein is the cell of embodiment 120, wherein the first and second polypeptides are at least 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5% identical, or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 86-124 or 2044-2070.
  • embodiment 122 provided herein is the cell of any one of embodiments 115 through 119, wherein the first and second polypeptides comprise a CAR or portion thereof that binds a binding partner comprising B7H3, BCMA, GPRC5D, CD8, CD8a, CD20, CD22, CD28, 4-1BB, or CD3zeta or a portion thereof.
  • the first and second polypeptides are at least 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5% identical, or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 86- 104, 116-124, or 2044-2070.
  • embodiment 124 is the cell of any one of embodiments 115 through 123, wherein the first CAR or portion thereof binds to a binding partner different than the second CAR or portion thereof.
  • embodiment 125 provided herein is a composition comprising a pharmaceutically acceptable excipient and a cell of any one of embodiments 20 through 124.
  • composition 126 is a composition for editing a plurality of sites in the genome of a target cell, wherein for each integer x, the composition comprises: (a) a donor template (D)x comprising a polynucleotide encoding a polypeptide comprising a CAR or portion thereof (CAR)x; (b) a Type V nucleic acid-guided nuclease (N)x; (c) a guide nucleic acid (gNA)x; wherein (CAR)x is different for each integer.
  • (N)x comprises MAD7.
  • compositions 126 or 127 wherein (gNA)x comprises a dual gNA.
  • composition 129 provided herein is the composition of any one of embodiments 126 through 128, wherein (D)x comprises a polynucleotide encoding a polypeptide comprising a CAR or portion thereof that binds a binding partner comprising B7H3, BCMA, GPRC5D, CD8, CD8a, CD19, CD20, CD22, CD28, 4-1BB, or CD3zeta or a portion thereof.
  • composition of embodiment 129 wherein (D)x comprises a polynucleotide encoding a CAR or portion thereof that comprises a polypeptide at least 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5% identical, or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 86-124 or 2044-2070.
  • embodiment 132 provided herein is the composition of embodiment 132, wherein (D)x comprises a polynucleotide encoding a CAR or portion thereof that comprises a polypeptide at least 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5% identical, or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 86-104, 116-124, or 2044-2070.
  • embodiment 133 provided herein is the composition of any one of embodiments 126 through 132, wherein the number of different integers x is at least 2, 3, 4, 5, 6, 7, 8, or 9 and no more than 10, 9, 8, 6, 5, 4, or 3.
  • embodiment 134 provided herein is the composition of embodiment 133, where the number of different integers x is 2-10.
  • embodiment 135 provided herein is the composition of embodiment 134, wherein the number of different integers x is 2-5.
  • embodiment 136 provided herein is a method for editing a genome of a target cell comprising introducing a first composition of any one of embodiments 115 through 125 into target cell.
  • embodiment 137 provided herein is the method of embodiment 136, further comprising adding an additive that stabilizes the nucleic acid-guided nuclease complex to the first composition prior to introducing the composition into the cell.
  • embodiment 138 provided herein is the method of embodiment 136 or 137, wherein introducing comprises electroporation.
  • embodiment 139 provided herein is the method of any one of embodiments 136 through 138, further comprising adding an additive to a cell growth medium that reduces non-homologous end joining (NHEJ) before or after introducing the composition into the cell.
  • NHEJ non-homologous end joining
  • embodiment 140 provided herein is the method of any one of embodiments 136 through 139, further comprising expanding the cell after introducing the composition into the cell.
  • embodiment 141 provided herein is the method of any one of embodiments 136 through 139, further comprising differentiating the cell after introducing the composition into the cell and/or expanding.
  • embodiment 142 provided herein is the method of any one of embodiments 136 through 141, further comprising introducing a second composition comprising a composition of any one of embodiments 115 through 125, wherein the second composition is different from the first composition into the cell and/or progeny of the cell.
  • embodiment 143 provided herein is the method of embodiment 142, further comprising introducing a third composition comprising a composition of any one of embodiments 115 through 125, wherein the third composition is different from the first and/or second compositions into the cell and/or progeny of the cell. 1.
  • embodiment 144 provided herein is one or more progeny of the cell or cells of embodiment 9 or any one of embodiments 20 through 110. [0293] VIII. Examples A.
  • Example 1 Primary human pan T-cells were isolated from whole leukopaks, processed on the day of receipt, and CD3-positive pan T-cells were separated from other peripheral blood mononuclear cells. Cells were characterized by flow cytometry before and after negative selection for viability, CD3 expression, and CD4/CD8 positivity. Cells were gated for proper size/shape, and singlets were selected. Cells displayed >98% viability prior to and following enrichment for pan T-cells, and the negative selection strategy resulted in enrichment of CD3 positive cells from 76.8% to 97.0%. Additionally, the CD4:CD8 ratio was maintained through the enrichment. The cells were frozen and used as needed.
  • Viability was measured by imaging in a flow cell with a volume of 1.4 ⁇ L using the Nucleocounter NC-200 and Via1 cassettes after staining cells Acridine orange and DAPI to differentiate live cells (acridine orange positive cells) from dead cells (DAPI positive cells).
  • Primary human pan T-cell specific nucleofection conditions including nucleofection buffer, nucleofection program (EO-115), and IL-2 concentration (200 IU/mL), were obtained from recommendations by Lonza and Nucleofection solution.8-12% CAR expression for each of the two CARs was observed ( Figures 9A and B; 2 nd and 3 rd bars for single (FL gRNA) and dual (STAR) gRNAs respectively).
  • nucleofection program EH-115 included in the nucleofection reaction.
  • inclusion of a ssODN in the nucleofection reaction increased delivery of the gene-editing reagents in primary human pan T- cells.
  • inclusion of a 200 nt ssODN in the nucleofection solution yielded high viability at day 11 post-nucleofection and CAR expression up to 40% when using 1 ⁇ g linearized dsDNA (ldsPLA074).
  • Figure 3A shows editing efficiency for three simultaneously genomic modifications comprising triple knock-out (KO) of HLA-1, HLA-2, and TCR as measured by flow cytometry following three treatment conditions: (1) untreated control; (2) treatment with gRNAs comprising a single polynucleotide (FL gRNA) in the presence of linear double stranded DNA (ldsPLA074); (3) treatment with gRNAs comprising a dual guide RNA (STAR) in the presence of linear double stranded DNA; and (4) treatment with gRNAs comprising a dual guide RNA (STAR) in the presence of linear double stranded DNA using improved conditions as described above.
  • gRNAs comprising a single polynucleotide
  • ldsPLA074 linear double stranded DNA
  • STAR dual guide RNA
  • STAR dual guide RNA
  • Figure 9B shows editing efficiency for three simultaneously genomic modifications comprising triple knock-out (KO) of HLA-1, HLA-2, and TCR as well as insertion of a polynucleotide encoding a CAR polypeptide as measured as measured by flow cytometry following three treatment conditions: (1) untreated control; (2) treatment with gRNAs comprising a single polynucleotide (FL gRNA) in the presence of linear double stranded DNA (ldsPLA074); (3) treatment with gRNAs comprising a dual guide RNA (STAR) in the presence of linear double stranded DNA; and (4) treatment with gRNAs comprising a dual guide RNA (STAR) in the presence of linear double stranded DNA using improved conditions as described above.
  • gRNAs comprising a single polynucleotide
  • ldsPLA074 linear double stranded DNA
  • STAR dual guide RNA
  • STAR dual guide RNA
  • compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.
  • a cell includes a plurality of cells, including mixtures thereof. Where the plural form is used for compounds, salts, or the like, this is taken to mean also a single compound, salt, or the like.
  • the expression “at least one of” includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use.
  • the expression “and/or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.

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Abstract

Des systèmes CRISPR-Cas ont été modifiés à diverses fins, telles que le clivage d'ADN génomique, l'édition de base, l'édition d'épigénome et l'imagerie génomique. La présente invention divulgue des compositions, des méthodes et/ou des kits destinés à l'ingénierie de génomes de cellules humaines à l'aide de systèmes CRISPR-Cas pour comprendre un ou plusieurs polypeptides comprenant un ou plusieurs CAR ou des parties de ceux-ci.
EP23808207.7A 2022-05-16 2023-05-16 Compositions et méthodes d'ingénierie de cellules Pending EP4526333A2 (fr)

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