WO2026015461A1 - Systèmes crispr-cas de type i - Google Patents

Systèmes crispr-cas de type i

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Publication number
WO2026015461A1
WO2026015461A1 PCT/US2025/036667 US2025036667W WO2026015461A1 WO 2026015461 A1 WO2026015461 A1 WO 2026015461A1 US 2025036667 W US2025036667 W US 2025036667W WO 2026015461 A1 WO2026015461 A1 WO 2026015461A1
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seq
type
nucleic acid
sequence identity
cas
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Inventor
Yiping QI
Simon SRETENOVIC
Micah DAILEY
Joshua CLEM
Neena PYZOCHA
Thomas Dubois
Hannah DIRESTA
Gregory Peel
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University of Maryland College Park
Inari Agriculture Technology Inc
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University of Maryland College Park
Inari Agriculture Technology Inc
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    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • 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
    • 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
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • 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
    • 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

  • CRISPR systems generally include two genetic components: the CRISPR array composed of conserved direct repeats separated by unique spacers derived from invading DNA molecules, and Cas genes encoding Cas proteins. Cas proteins confer three molecular stages of defense: spacer acquisition, crRNA (CRISPR RNA) maturation, and target interference.
  • CRISPR RNA crRNA
  • the most well-known CRISPR technology is perhaps the CRISPR-Cas9 system, a class 2 type II CRISPR system that was first demonstrated to be a potent genome editing tool in 2012.
  • the CRISPR-Cas9 system is a single monomeric ribonucleoprotein complex of approximately 160 kDa in size. It is capable of introducing small-scale deletions or nicks in a targeted manner.
  • Class 1 Type I CRISPR systems although more abundant in prokaryotes as compared to Class 2 Type II CRISPR systems, have been largely overlooked as genome editing tools due to their large, multidomain ribonucleoprotein complexes.
  • the identification of novel Type I CRIPSR systems is useful 9582-111817-02 for the development of new tools for various biotechnology applications, particularly for the introduction of large-scale deletions, which is not achieved using CRISPR-Cas9 systems.
  • SUMMARY Type I CRISPR-Cas systems include various Cas proteins that form a CRISPR associated complex for antiviral defense (CASCADE).
  • CASCADE Binding of CASCADE to a target sequence recruits Cas3 (or Cas2-Cas3), which harbors endonuclease and helicase activity.
  • the CASCADE complex can also be engineered to be fused to a non-specific endonuclease, for example, a Fok1 endonuclease domain, to achieve targeted nuclease activity without the need of a Cas2-Cas3, Cas3, or other Cas nuclease.
  • Type I systems can be divided into six subtypes spanning from IA to IG with each harboring a distinct Cas8 homolog.
  • Novel Class 1 Type IC and Type IF CRISPR systems have the potential to be used, for example, as biotechnological tools for facilitating genetic or epigenetic modifications, and/or for modulating transcription of a target gene.
  • a bioinformatics approach was taken to identify novel Class 1 Type IC and Type IF CRISPR systems sourced from bacteria.
  • the list of candidates was narrowed based on the presence of a full-size CRISPR locus (as defined for each subtype in the scientific literature), predicted activity at room temperature, and simple predicted respective protospacer adjacent motifs (PAMs). With this selection criteria, the list was narrowed to 25 top candidates.
  • novel class 1 type I CRISPR-Cas systems including a CRISPR-associated complex for antiviral defense (CASCADE) that includes a plurality of Cas proteins, and an artificial guide RNA (gRNA).
  • the artificial gRNA is not, or otherwise excludes, a naturally occurring gRNA.
  • the CASCADE complex is from: i) a Methylomonas methanica type IF system CASCADE complex; ii) a Sphingobium amiense type IC system CASCADE complex; iii) an Alteromonas mediterranea type IFv system CASCADE complex; or iv) a Pragia fontium type IF system CASCADE complex.
  • the Methylomonas methanica type IF system CASCADE complex includes a Methylomonas methanica Cas5f, Cas6, Cas7f, and/or Cas8f.
  • the Sphingobium amiense type IC system CASCADE complex includes a Sphingobium amiense Cas5, Cas7, Cas8c, and/or Cas11.
  • the Alteromonas mediterranea type IFv system CASCADE complex includes an Alteromonas mediterranea Cas5fv, Cas6, and/or Cas7fv.
  • the Pragia fontium type IF system CASCADE complex includes a Pragia fontium Cas5f, Cas6, Cas7f, and/or Cas8f.
  • a class 1 type I CRISPR-Cas system disclosed herein further comprises a Cas3, Cas2-Cas3, or Fok1 nuclease domain.
  • an effector molecule e.g., a transcriptional activator, a transcriptional repressor, a base editor, a reporter, a nuclear localization signal (NLS), etc.
  • a base 9582-111817-02 editor e.g., cytidine deaminase or adenine deaminase
  • Cas proteins disclosed herein thereby linked to a CASCADE complex disclosed herein.
  • recombinant nucleic acids encoding one or more components of a class 1 type I CRISPR-Cas system disclosed herein.
  • the recombinant nucleic acid encodes one or more of SEQ ID NOs: 1-19.
  • the recombinant nucleic acid encodes two or more of SEQ ID NOs: 1-19.
  • the recombinant nucleic acid includes one or more of SEQ ID NOs: 20-38, or a degenerate variant thereof.
  • the recombinant nucleic acid includes two or more of SEQ ID NOs: 20-38, or a degenerate variant thereof.
  • the recombinant nucleic acids are codon optimized for expression in a host cell.
  • vectors including a recombinant nucleic acid disclosed herein, and host cells including a class 1 type I CRISPR-Cas system disclosed herein, or a recombinant nucleic acid or vector disclosed herein.
  • methods of modifying a target nucleic acid sequence or altering expression of a target nucleic acid in a cell including introducing a class 1 type I CRISPR-Cas system disclosed herein into the cell.
  • introducing the class 1 type I CRISPR-Cas system into the cell includes transforming the cell with a nucleic acid (including a vector) encoding the class 1 type I CRISPR-Cas system, or transforming a ribonucleoprotein (RNP) particle including the class 1 type I CRISPR-Cas system.
  • the cell is a plant cell. Further provided are plants regenerated from cells transformed with nucleic acids, vectors, or RNPs disclosed herein.
  • FIGS.1A-1B show Sphingobium amiense DSM16289 type IC system bioinformatic characterization and PAM prediction.
  • FIGS.2A-2B show Methylomonas methanica MC09 type IF system bioinformatic characterization and PAM prediction.
  • FIGS.3A-3B show Pragia fontium 24613 type IF system bioinformatic characterization and PAM prediction.
  • FIGS.4A-4B show Alteromonas mediterranea CP48/49 type IF system bioinformatic characterization and PAM prediction.
  • FIGS.5A-5C show the preparation of a single T-DNA vector encoding Cas3, CASCADE and gRNA.
  • FIG.6 shows a schematic representation of PCR amplicon preparation in relation to a target site.
  • FIG.7 shows nanopore sequencing results of Methylomonas methanica MC09 – type IF biological replicate III at OsGS3-gR1 target site in rice protoplasts.
  • FIG.8 shows nanopore sequencing results of Methylomonas methanica MC09 – type IF biological replicate IV at OsGS3-gR1 target site in rice protoplasts.
  • FIG.9 shows agarose gel results of long-range nested PCR at OsGS3 and OsGW2 target sites. Arrows note putative deletions and asterisks note WT amplicons.
  • FIGS.10A-10B show IGV coverage of two transgenic events at two target sites (FIG.10A) and genome editing summary statistics (FIG.10B).
  • FIGS.11A-11D show editing efficiency (FIG.11A) and deletion size and frequency (FIGS. 11B-11D) for the two target sites (OsGS3 and OsGW2) based on SIQ output.
  • SEQUENCES The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and single letter code for amino acids, in compliance with 37 C.F.R. ⁇ 1.831-1.834.
  • SEQ ID NO: 1 is an exemplary amino acid sequence of Cas5f from a Methylomonas methanica type IF system.
  • SEQ ID NO: 2 is an exemplary amino acid sequence of Cas6 from a Methylomonas methanica type IF system.
  • SEQ ID NO: 3 is an exemplary amino acid sequence of Cas7f from a Methylomonas methanica type IF system.
  • SEQ ID NO: 7 is an exemplary amino acid sequence of Cas7 from a Sphingobium amiense type IC system.
  • SEQ ID NO: 8 is an exemplary amino acid sequence of Cas8c from a Sphingobium amiense type IC system.
  • SEQ ID NO: 10 is an exemplary amino acid sequence of Cas3 from a Sphingobium amiense type IC system.
  • SEQ ID NO: 13 is an exemplary amino acid sequence of Cas7fv from an Alteromonas mediterranea type IFv system.
  • SEQ ID NO: 14 is an exemplary amino acid sequence of Cas2-Cas3 from an Alteromonas mediterranea type IFv system.
  • MNVLILPHINIHNANALSSSFTIGFPAMTAWLGFVHALERKLNQAGLPELMLHSAAVVSHRC DVQTHKGEGDFVHSIIGTGNPLDKDGSRSAFIEEARCHLDVSLVIEWGGNEDQVQHADFAEQ LQAVIATMKVAGGDVLSMHRPLNQSVDIDNPQETRALLRKLMPGYVLIERRDLMTEAMAQ GSDALDALLSYLTVNHRCEQLEDGSVIWRSQRKASGWIVPIATGFQGISPLGEAKNQRDPSVP HRFAESVVTLGEFVMAHKIQHLDDMLWHYHNDLENDLYLCQQVNAINEHQ SEQ ID NO: 16 is an exemplary amino acid sequence of Cas6(Csy4) from a Pragia fontium type IF system.
  • SEQ ID NO: 17 is an exemplary amino acid sequence of Cas7f(Csy3) from a Pragia fontium type IF system.
  • SEQ ID NO: 29 is an exemplary nucleic acid encoding Cas3 from a Sphingobium amiense type IC system.
  • CTTAGAAA SEQ ID NO: 40 is an exemplary Methylomonas methanica type IF 3’ mature direct repeat.
  • GTCCGCCGCCGCACAGGCGG SEQ ID NO: 41 is an exemplary Methylomonas methanica type IF self-processing direct repeat.
  • GTCCGCCGCCGCACAGGCGGCTTAGAAA SEQ ID NO: 42 is an exemplary Alteromonas mediterranea type IFv mature direct repeat.
  • CTTAGAAA SEQ ID NO: 43 is an exemplary Alteromonas mediterranea type IFv 3’ mature direct repeat.
  • GTTCATGGCCGCACAGGCCA SEQ ID NO: 44 is an exemplary Alteromonas mediterranea type IFv self-processing direct repeat.
  • GTTCATGGCCGCACAGGCCACTTAGAAA SEQ ID NO: 45 is an exemplary Sphingobium amiense type IC 5’ mature direct repeat.
  • TGGATCGAAAC SEQ ID NO: 46 is an exemplary Sphingobium amiense type IC 3’ mature direct repeat.
  • GTCGCCTCCCACACGGAGGCG SEQ ID NO: 47 is an exemplary Sphingobium amiense type IC self-processing direct repeat.
  • CRISPRs clustered regularly interspaced short palindromic repeats
  • CRISPR-based systems can be developed to provide powerful tools for targeted gene editing or epigenomic editing, as well as targeted modulation of gene expression.
  • Class II CRISPR systems are the most well characterized and rely on a single Cas protein (e.g., Cas9, Cas12, or Cas13).
  • class 1 type I CRISPR systems rely on a complex of Cas proteins termed “CRISPR-associated complex for antiviral defense” (CASCADE), which recruit an endonuclease (e.g., Cas3 or Cas2-Cas3) upon binding to a target nucleic acid.
  • Class 1 type I systems typically include a combination of three or more of: Cas4, Cas5, Cas6, Cas7, Cas8 (Cse1), and Cas11 (Cse2). Only one unit of each Cas is required for Cascade assembly with the exception of Cas7.
  • Cas7 units are present and scalable depending on the size of the spacer region.
  • there are six Cas7 proteins in a CASCADE complex however, the number of Cas7 proteins recruited to an R-loop is proportional to the length of the crRNA spacer sequence, with one Cas7 recruited for 9582-111817-02 every 6 nucleotides (nt) (see, e.g., Tuminauskaite et al., “DNA interference is controlled by R-loop length in a type I-F1 CRISPR-Cas system,” BMC Biology 18:65, 2020).
  • nt nucleot
  • control is a historical control or a standard reference or range (a typical measurement or range observed for a particular population, such as a typical measurement (e.g., gene expression) or range for an unmodified host cell).
  • Expression (of a nucleic acid) includes transcription and/or translation of the nucleic acid.
  • Expression Cassette A nucleic acid fragment designed for expression of a particular gene (or genes) in a host cell. Expression cassettes can be included in a vector. An expression cassette can include regulatory elements, such as promoters and/or terminators.
  • nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids. Absolute purity or isolation is not required, it is intended as a relative term.
  • a purified/isolated protein, nucleic acid, or cell preparation is one in which the protein, nucleic acid, or cell is more enriched than the protein, nucleic acid, or cell is in its initial environment.
  • a preparation is purified/isolated such that the protein, nucleic acid, or cell represents at least 50% of the total content of the preparation.
  • a substantially purified protein or nucleic acid is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% pure. Thus, in one specific, non-limiting example, a substantially purified protein or nucleic acid is 90% free of other components.
  • Modified Cell or Plant A cell or plant that includes an artificial genetic modification. Modified cells or plants are not naturally occurring. In some aspects, a modified cell or plant is a genome-edited cell or plant (e.g., by CRISPR-based editing). In other aspects, a modified cell or plant is a transgenic plant (a plant that includes a transgene).
  • a nucleic acid sequence is “operably linked” when it is placed in a functional relationship with a second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, are in the same reading frame.
  • Promoter A nucleic acid control sequence that directs transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription.
  • a promoter also optionally includes distal enhancer or repressor elements.
  • a “constitutive promoter” is a promoter that is continuously active and is not subject to regulation by external signals or molecules. 9582-111817-02 In contrast, the activity of an “inducible promoter” is regulated by an external signal or molecule (for example, a transcription factor).
  • nucleic acid disclosed herein is operably linked to a pol III promoter (e.g., U6), a pol II promoter, ubiquitin promoter, Cauliflower Mosaic Virus (CaMV) 35S promoter, or RUBISCO promoter.
  • Recombinant A nucleic acid or protein that has a sequence made by an artificial combination of two otherwise separated segments of sequence (e.g., a “chimeric” sequence).
  • a recombinant protein includes a protein produced from a recombinant nucleic acid.
  • Regulatory Element A term that includes promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).
  • the reporter protein is positioned downstream of and in frame with a gene of interest, such that the reporter protein is co-expressed with the gene of interest.
  • Sequence Identity The degree of similarity between amino acid or nucleic acid sequences. Sequence identity is frequently measured in terms of percentage identity (or percent identity); the higher the percentage, the more similar the two sequences are. Homologs of a polypeptide (or nucleotide sequence) will possess a relatively high degree of sequence identity when aligned using standard methods. Methods of alignment of sequences for comparison have been described.
  • BLAST Basic Local Alignment Search Tool
  • Various types of BLAST are available, for example, blastp, blastn, blastx, tblastn and tblastx.
  • a description of how to determine sequence identity using this program is available on the NCBI website and other resources.
  • percent sequence identity is determined by using BLAST with default parameters.
  • Transformed A transformed cell is a cell into which an exogenous biological macromolecule (e.g., DNA, RNA, protein, ribonucleoprotein (RNP), etc.) has been introduced by a molecular biology technique.
  • an exogenous biological macromolecule e.g., DNA, RNA, protein, ribonucleoprotein (RNP), etc.
  • the vector is a transfer DNA (T-DNA) vector suitable for agrobacterium-mediated transformation or a vector suitable for biolistics.
  • T-DNA transfer DNA
  • gRNA artificial guide RNA
  • a class 1 type I CRISPR-Cas system disclosed herein further includes an endonuclease, for example, a Cas3, Cas2-Cas3, or Fok1 nuclease.
  • the CASCADE complex is a complex from Methylomonas methanica, Sphingobium amiense, Alteromonas mediterranea, or Pragia fontium.
  • the CASCADE complex is from a Methylomonas methanica type IF system.
  • the CASCADE complex is from a Sphingobium amiense type IC system.
  • the CASCADE complex is from an Alteromonas mediterranea type IFv system.
  • the CASCADE complex is from a Pragia fontium type IF system.
  • the CASCADE complex includes a plurality of Cas proteins, for example, at least 2 Cas proteins, at least 3 Cas proteins, or at least 4 Cas proteins. In some aspects, the CASCADE complex includes at least 3 Cas proteins. In some aspects, the plurality of Cas proteins includes one or more of: Cas5 or a homolog thereof, Cas6 or a homolog thereof, Cas7 or a homolog thereof, Cas8 or a homolog thereof, and Cas11 or a homolog thereof.
  • the plurality of Cas proteins includes two or more of: Cas5 or a homolog thereof, Cas6 or a homolog thereof, Cas7 or a homolog thereof, Cas8 or a homolog thereof, and Cas11 or a homolog thereof. In some aspects, the plurality of Cas proteins includes three or more of: Cas5 or a homolog thereof, Cas6 or a homolog thereof, Cas7 or a homolog thereof, Cas8 or a homolog thereof, and Cas11 or a homolog thereof. In some aspects, the CASCADE complex includes a plurality of Cas7 or a homolog thereof (e.g., 3-10, 3-8, 5-7, or 6).
  • the CASCADE complex includes one or more of: Cas5 or a homolog thereof, Cas6 or a homolog thereof, Cas8 or a homolog thereof, and/or Cas11 or a homolog thereof, and a plurality of Cas7 or a homolog thereof (e.g., 3-10, 3-8, 5-7, or 6).
  • the CASCADE complex includes (i) two or more of: Cas5 or a homolog thereof, Cas6 or a homolog thereof, Cas8 or a homolog thereof, and/or Cas11 or a homolog thereof, and (ii) a plurality of Cas7 or a homolog thereof (e.g., 3-10, 3-8, 5-7, or 6).
  • the CASCADE complex includes one or more of: Cas5, Cas5f, Cas5fv, Cas6, Cas7, Cas7f, Cas7fv, Cas8c, Cas8f, and Cas11. In some aspects, the CASCADE complex includes two or more of: Cas5, Cas5f, Cas5fv, Cas6, Cas7, Cas7f, Cas7fv, Cas8c, Cas8f, and Cas11. In some aspects, the CASCADE complex includes three or more of: Cas5, Cas5f, Cas5fv, Cas6, Cas7, Cas7f, Cas7fv, Cas8c, Cas8f, and Cas11.
  • the CASCADE complex includes Cas5, Cas5f, or Cas5fv. In some aspects, the CASCADE complex includes Cas6. In some aspects, the CASCADE complex includes Cas7, Cas7f, or Cas7fv. In some aspects, the CASCADE complex 9582-111817-02 includes Cas8c or Cas8f. In some aspects, the CASCADE complex includes Cas11. In some aspects, a CASCADE complex disclosed herein includes a plurality of Cas7, Cas7f, and/or Cas7fv.
  • the CASCADE complex includes one or more of: Cas5, Cas5f, Cas5fv, Cas6, Cas8c, Cas8f, and/or Cas11, and a plurality of Cas7, Cas7f, or Cas7fv (e.g., 3-10, 3-8, 5-7, or 6).
  • the CASCADE complex includes (i) two or more of: Cas5, Cas5f, Cas5fv, Cas6, Cas8c, Cas8f, and/or Cas11, and (ii) a plurality of Cas7, Cas7f, or Cas7fv (e.g., 3-10, 3-8, 5-7, or 6).
  • the CASCADE complex includes (i) three or more of: Cas5, Cas5f, Cas5fv, Cas6, Cas8c, Cas8f, and/or Cas11, and (ii) a plurality of Cas7, Cas7f, or Cas7fv (e.g., 3-10, 3-8, 5-7, or 6).
  • the CASCADE complex includes: Cas5f, Cas6, Cas7f, and/or Cas8f.
  • the CASCADE complex includes: Cas5, Cas7, Cas8c, and/or Cas11.
  • the CASCADE complex includes or consists of: Cas5f, Cas6, and/or Cas8f, and a plurality of Cas7f (e.g., 3-10, 3-8, 5-7, or 6).
  • the CASCADE complex includes a Cas protein that has at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) sequence identity to one or more of SEQ ID NOS: 1-4, 6-9, 11-13, or 15-18.
  • the CASCADE complex includes a Cas protein that has at least 95% sequence identity to one or more of SEQ ID NOS: 1-4, 6- 9, 11-13, or 15-18. In some aspects, the CASCADE complex includes a Cas protein that includes or consists of any one of SEQ ID NOS: 1-4, 6-9, 11-13, or 15-18. In some aspects, the CASCADE complex includes a Cas protein that has at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 100%) sequence identity to each of SEQ ID NOs: 1, 2, 3, and 4, respectively.
  • the CASCADE complex includes a Cas protein that has at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 100%) sequence identity to each of SEQ ID NOs: 6, 7, 8, and 9, respectively.
  • the CASCADE complex includes a Cas protein that has at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 100%) sequence identity to each of SEQ ID NOs: 11, 12, and 13, respectively.
  • the CASCADE complex includes a Cas protein that has at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 100%) sequence identity to each of SEQ ID NO: 15, 16, 17, and 18, respectively.
  • the CASCADE complex includes a Cas protein comprising each of SEQ ID NOs: 1, 2, 3, and 4, respectively.
  • the 9582-111817-02 CASCADE complex includes a Cas protein comprising each of SEQ ID NOs: 6, 7, 8, and 9, respectively.
  • the CASCADE complex includes a Cas protein comprising each of SEQ ID NOs: 11, 12, and 13, respectively.
  • the CASCADE complex includes a Cas protein comprising each of SEQ ID NOs: 15, 16, 17, and 18, respectively.
  • the CASCADE complex is a Methylomonas methanica type IF system CASCADE complex.
  • the Methylomonas methanica type IF system CASCADE complex includes a Methylomonas methanica Cas5f, Cas6, Cas7f, and/or Cas8f. In some aspects, the Methylomonas methanica type IF system CASCADE complex includes or consists of Methylomonas methanica Cas5f, Cas6, Cas7f, and Cas8f.
  • a class 1 type I CRISPR-Cas system including a Methylomonas methanica type IF system CASCADE complex further includes an endonuclease, for example Methylomonas methanica Cas2-Cas3 or a Fok1 endonuclease domain.
  • the Methylomonas methanica type IF system CASCADE complex includes a Cas5f that has at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 100%) sequence identity to SEQ ID NO: 1; a Cas6 that has at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 100%) sequence identity to SEQ ID NO: 2; a Cas7f that has at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 100%) sequence identity to SEQ ID NO: 3; and/or a Cas8f that has at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 100%) sequence identity to SEQ ID NO: 4.
  • a Cas5f that has at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 100%) sequence identity to SEQ ID NO: 1
  • a Cas6 that has at least
  • the Methylomonas methanica type IF system CASCADE complex includes a Cas5f that has at least 95% sequence identity to SEQ ID NO: 1; a Cas6 that has at least 95% sequence identity to SEQ ID NO: 2; a Cas7f that has at least 95% sequence identity to SEQ ID NO: 3; and/or a Cas8f that has at least 95% sequence identity to SEQ ID NO: 4.
  • the Methylomonas methanica type IF system CASCADE complex includes a Cas5f that includes or consists of SEQ ID NO: 1; a Cas6 that includes or consists of SEQ ID NO: 2; a Cas7f that includes or consists of SEQ ID NO: 3; and/or a Cas8f that includes or consists of SEQ ID NO: 4.
  • the Methylomonas methanica type IF system CASCADE complex includes a Cas5f that includes or consists of SEQ ID NO: 1; a Cas6 that includes or consists of SEQ ID NO: 2; a Cas7f that includes or consists of SEQ ID NO: 3; and a Cas8f that includes or consists of SEQ ID NO: 4.
  • the Methylomonas methanica type IF system CASCADE complex consists of a Cas5f that includes or consists of SEQ ID NO: 1; a Cas6 that includes or consists of SEQ ID NO: 2; a Cas7f that includes or consists of SEQ ID NO: 3; and a Cas8f that includes or consists of SEQ ID NO: 4.
  • a class 1 type I CRISPR-Cas system including a Methylomonas methanica type IF system CASCADE complex disclosed herein further includes a Methylomonas methanica Cas2-Cas3.
  • the Cas2-Cas3 that has at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 100%) sequence identity to SEQ ID NO: 5. In some aspects, the Cas2-Cas3 has at least 95% sequence identity to SEQ ID NO: 5. In some aspects, the Cas2-Cas3 includes or consists of SEQ ID NO: 5. In some aspects, a class 1 type I CRISPR-Cas system including a Methylomonas methanica type IF system CASCADE complex disclosed herein further includes a Fok1 nuclease domain.
  • the Fok1 nuclease domain has at least 80% (e.g., at least 85%, 90%, 95%, 98%, or 100%) sequence identity to SEQ ID NO: 102. In some aspects, the Fok1 nuclease domain has at least 95% sequence identity to SEQ ID NO: 102. In some aspects, the Fok1 nuclease domain includes or consists of SEQ ID NO: 102.
  • the Pragia fontium type IF system CASCADE complex includes a Cas5f that includes or consists of SEQ ID NO: 15; a Cas6 that includes or consists of SEQ ID NO: 16; a Cas7f that includes or consists of SEQ ID NO: 17; and/or a Cas8f that includes or consists of SEQ 9582-111817-02 ID NO: 18.
  • the Pragia fontium type IF system CASCADE complex includes a Cas5f that includes or consists of SEQ ID NO: 15; a Cas6 that includes or consists of SEQ ID NO: 16; a Cas7f that includes or consists of SEQ ID NO: 17; and a Cas8f that includes or consists of SEQ ID NO: 18.
  • the Pragia fontium type IF system CASCADE complex consists of a Cas5f that includes or consists of SEQ ID NO: 15; a Cas6 that includes or consists of SEQ ID NO: 16; a Cas7f that includes or consists of SEQ ID NO: 17; and a Cas8f that includes or consists of SEQ ID NO: 18.
  • Non-limiting examples of synthetic amino acids include: aminocyclohexane carboxylic acid, norleucine, a-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4- hydroxyproline, 4- aminophenylalanine, 4- nitrophenylalanine, 4-chlorophenylalanine, 4- carboxyphenylalanine, ⁇ -phenylserine ⁇ -hydroxyphenylalanine, phenylglycine, ⁇ -naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1 ,2,3,4- tetrahydroisoquinoline-3- carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, ⁇ ', ⁇ '-dibenzyl-lysine, 6-
  • an effector molecule is linked to a Cas protein disclosed herein.
  • the effector molecule is a transcriptional activator, a transcriptional repressor, chromatin remodeling protein, histone modifying protein (e.g., histone acetyltransferases (HATs), histone deacetylases, histone methyltransferases, histone kinases, histone ubiquitinases, etc.), a base editor, a reporter, a tag, a nuclear localization signal (NLS), or a combination of two or more thereof.
  • HATs histone acetyltransferases
  • NLS nuclear localization signal
  • chromatin remodeling or histone modifying proteins include, but are not limited to DNA methyltransferases (DNMTs), DNA demethylation enzymes (e.g., TET1, TET2 and TET3), histone acetyltransferases CBP and p300, and histone methyltransferases (SMYD3, PRDM9 and DOT1L).
  • DNMTs DNA methyltransferases
  • TET1, TET2 and TET3 DNA demethylation enzymes
  • histone acetyltransferases CBP and p300 histone acetyltransferases
  • SYD3, PRDM9 and DOT1L histone methyltransferases
  • Exemplary suitable base editors include, but are not limited to, cytidine deaminases, adenosine deaminases, or dual base editing deaminases that confer simultaneous C-to-T and A-to-G base editing.
  • a Cas protein disclosed herein is linked to a cytidine deaminase or adenine deaminase (see, e.g., Zimmermann et al. “A cas3-base editing tool for targetable in vivo mutagenesis” Nature Communications, 14:3389, 2023).
  • Exemplary suitable nuclear localization signals include, but are not limited to simian virus 40 (SV40) and nucleoplasmin NLS.
  • Exemplary bioluminescent reporters include, for example, luciferase (e.g., Renilla luciferase).
  • Exemplary colorimetric reporters include, but are not limited to, LacZ, GusA, CelB, AES, NagZ, PhoA, and Est2.
  • the reporter is an enzyme, such as luciferase, horseradish peroxidase, alkaline phosphatase, LacZ, GusA, CelB, AES, NagZ, PhoA, or Est2.
  • an enzyme reporter produces a detectable signal upon contact with an enzymatic substrate.
  • a tag includes any molecule that facilitates capture/protein isolation, and/or detection.
  • the tag is a 6X histidine tag, glutathione-S-transferase (GST) tag, IgG-Fc tag, maltose- binding protein tag, FLAG tag, or biotin tag.
  • the tag is a protein for which antibodies are available and which can be detected or used to facilitate protein purification.
  • the tag binds to a protein purification substrate or resin, such as a substrate or resin including protein A or/or protein G (e.g., protein A and/or G Sepharose® (crosslinked agarose bead), or magnetic beads including protein A and/or G), glutathione resin, streptavidin resin, or other affinity resin.
  • a Cas protein When a Cas protein is part of a fusion protein, the starting methionine (methionine in position 1 of an amino acid sequence) can be deleted.
  • a Cas protein disclosed herein e.g., any of SEQ ID NOs: 1-19
  • an effector and Cas protein are joined by an amino acid linker.
  • the linker is an amino acid linker (such as 1-100 amino acids, such as 1-20 amino acids, 10- 9582-111817-02 30 amino acids, 20-40 amino acids, 30-50 amino acids, 40-60 amino acids, 50-70 amino acids, 60-80 amino acids, 70-90 amino acids, or 80-100 amino acids).
  • the effector molecule When the effector molecule is not a protein, the effector may be directly attached to the Cas protein by a non-peptide bond, for example, a thiol or amine bond.
  • Non-protein effector molecules may be attached to a Cas protein at any suitable location (e.g., a location where attachment can occur and does not significantly disrupt function of the Cas protein or the effector molecule), and are not limited to linkage at the N- or C-terminus.
  • an effector molecule can be linked to a Cas protein by a streptavidin-biotin linker.
  • the C-terminus of streptavidin is directly fused (for example, by a peptide bond) to the N-terminus of a Cas protein.
  • the C-terminus of a Cas protein is directly fused (for example by a peptide bond) to the N-terminus of streptavidin.
  • the Cas protein can also be indirectly linked to streptavidin, for example by a peptide linker, as discussed above with respect to effector molecules.
  • the effector molecule is biotinylated, and the Cas protein and the effector molecule are linked by the interaction of between streptavidin and biotin.
  • the Cas protein is biotinylated and the effector molecule is linked to a streptavidin moiety.
  • the class 1 type I CRISPR-Cas systems disclosed herein include an artificial guide RNA (gRNA).
  • the target site also known as the protospacer, has 100% complementarity with the CRISPR RNA spacer (the RNA component which surveys genomic DNA for complementarity).
  • the CRISPR RNA spacer the RNA component which surveys genomic DNA for complementarity.
  • the most abundant spacer lengths were observed to be 32 nucleotides for Type IF systems (IF-MM (Methylomonas methanica), IF-PF (Pragia fontium), and IF-AM (Alteromonas mediterranea)) and 34 nucleotides for Type IC-SA (Sphingobium amiense).
  • MS2 hairpins can be used to recruit MCP-transcriptional activator fusion proteins to a target sequence.
  • Other RNA aptamers may also be used, for example, PP7, Com, and BoxB.
  • Nucleic Acids, Vectors, and Host Cells Also disclosed are recombinant nucleic acid molecules encoding at least one Cas protein disclosed herein (e.g., any of SEQ ID NOs: 1-19).
  • the nucleic acid molecules disclosed herein can be, for example, DNA, RNA, cDNA, or synthetic derivatives thereof.
  • a vir helper plasmid (e.g., EHA101, EHA105, AGL-1, LBA4404, and GV2260), contains the vir genes of the Ti plasmid of Agrobacterium, and is co-transformed with a T-DNA vector to facilitate transfer and integration of a DNA of interest.
  • the T-DNA vector is suitable for biolistic delivery into a host cell.
  • a nucleic acid disclosed herein is directly introduced into a host cell (e.g., a plant cell) by biolistic-based transformation (e.g., gene gun).
  • sibirica asparagus (Asparagus officinalis), bananas (Musa spp.), barley (Hordeum vulgare), beans (Phaseolus spp.), blueberries and cranberries (Vaccinium spp.), cacao (Theobroma cacao), canola and rapeseed or oilseed rape, (Brassica napus), carnation (Dianthus caryophyllus), carrots (Daucus carota sativus), cassava (Manihot esculentum), cherry (Prunus avium), chickpea (Cider arietinum), chicory (Cichorium intybus), chili peppers and other capsicum peppers (Capsicum annuum, C.
  • Coffea arabica and Coffea canephora including Coffea arabica and Coffea canephora), cotton (Gossypium hirsutum), cowpea (Vigna unguiculata), cucumber (Cucumis sativus), currants and gooseberries (Ribes spp.), eggplant or aubergine (Solanum melongena), eucalyptus (Eucalyptus spp.), flax (Linum usitatissumum), geraniums (Pelargonium spp.), grapefruit (Citrus x paradisi), grapes (Vitus spp.) including wine grapes (Vitus vinifera), guava (Psidium guajava), irises (Iris spp.), lemon (Citrus limon), lettuce (Lactuca sativa), limes (Citrus spp.), maize (Zea mays), mango (Mangifera indica), mangos
  • the vector can also include one or more tags for purification of an encoded protein (e.g., a Cas protein disclosed herein), such as histidine (His), chitin- binding protein (CBP), maltose-binding protein (MBP), or glutathione-S-transferase (GST), or a streptavidin tag.
  • an encoded protein e.g., a Cas protein disclosed herein
  • His histidine
  • CBP chitin- binding protein
  • MBP maltose-binding protein
  • GST glutathione-S-transferase
  • streptavidin tag e.g., a streptavidin tag.
  • the vector further encodes a heat shock protein, molecular chaperone, or cofactor.
  • all of the Cas genes required for a class 1 type I CASCADE complex, an endonuclease (e.g., Cas3, Cas2- Cas3, or Fok1 endonuclease domain), and a gRNA are encoded in a single contiguous nucleic acid molecule that is transformed into a cell.
  • one or more components of a CASCADE complex disclosed herein are introduced by transforming a host cell with isolated protein, for example, isolated Cas proteins that assemble a CASCADE complex once in vivo, or an in vitro assembled CASCADE complex.
  • the host cell that has been or will be transformed with the Cas protein or CASCADE complex
  • the host cell can be engineered to express an artificial gRNA, or the artificial guide RNA can be directly transformed as RNA.
  • a gRNA is transformed as RNA, it does not need to be expressed by the host cell.
  • a class 1 type I CRISPR-Cas system disclosed herein is introduced into a host cell by transforming the cell with an isolated ribonucleoprotein (RNP) complex, which includes an assembled CASCADE complex associated with the artificial gRNA.
  • RNP ribonucleoprotein
  • Components of a CASCADE complex e.g., Cas proteins
  • a class 1 type I CRISPR-Cas system e.g., Cas proteins or gRNA
  • a gRNA is introduced into a cell before, after, or substantially at the same time as a CASCADE complex disclosed herein.
  • a native genomic sequence is replaced by the repair template sequence, thus introducing an insertion, deletion, or substitution into the genome.
  • the methods disclosed herein do not include introducing a repair template.
  • the cell may repair DNA breaks through endogenous mechanisms, which typically results in deletions or insertions.
  • Altering expression includes, for example, increasing or decreasing expression of a target nucleic acid.
  • expression is increased, for example, by targeting a transcriptional activator to a target sequence.
  • expression is decreased, for example, by targeting a transcriptional repressor or by introducing insertions or deletions that disrupt or abolish transcription of a target nucleic acid.
  • expression is increased or decreased, for example, by targeting a chromatin remodeling or histone modifying protein (e.g., histone acetyltransferases (HATs), deacetylases, methyltransferases, kinases, ubiquitinases, etc.) to a target sequence, thereby inducing epigenic changes that modulate gene expression.
  • a chromatin remodeling or histone modifying protein e.g., histone acetyltransferases (HATs), deacetylases, methyltransferases, kinases, ubiquitinases, etc.
  • HATs histone acetyltransferases
  • methyltransferases e.g., methyltransferases
  • kinases e.g., kinases, ubiquitinases, etc.
  • Recruitment of transcriptional activators, repressors, chromatin remodeling proteins, or histone modifying proteins can be achieved, for example,
  • a gRNA that includes an RNA binding motif, for example, an MS2 hairpin.
  • the cell is a bacterial, archaea, insect (for example, Spodoptera frugiperda cells), fungi (for example, yeast), plant, or animal cell (for example, mammalian cells, such as human cells).
  • suitable cells include, but are not limited to, Escherichia coli (E.
  • the cell is a plant cell (including protoplasts). In some aspects, the cell is in an isolated plant cell or part of an isolated plant tissue and the method is performed in vitro. In other aspects, the plant cell is not an isolated plant cell, for example, the plant cell is located in an intact plant or in a plant part or tissue. In such aspects, the method is performed in situ or in planta (e.g., agrobacterium-mediated transformation via floral dip).
  • the plant cell can be from a dicot or a monocot.
  • the plant cell is from or is part of a row crop plant, fruit-producing plant, tree, vegetable, or ornamental plant, including ornamental flowers, shrubs, trees, groundcovers, and turf grasses.
  • sibirica asparagus (Asparagus officinalis), bananas (Musa spp.), barley (Hordeum vulgare), beans (Phaseolus spp.), blueberries and cranberries (Vaccinium spp.), cacao (Theobroma cacao), canola and rapeseed or oilseed rape, (Brassica napus), carnation (Dianthus caryophyllus), carrots (Daucus carota sativus), cassava (Manihot esculentum), cherry (Prunus avium), chickpea (Cider arietinum), chicory (Cichorium intybus), chili peppers and other capsicum peppers (Capsicum annuum, C.
  • Coffea arabica and Coffea canephora including Coffea arabica and Coffea canephora), cotton (Gossypium hirsutum), cowpea (Vigna unguiculata), cucumber (Cucumis sativus), currants and gooseberries (Ribes spp.), eggplant or aubergine (Solanum melongena), eucalyptus (Eucalyptus spp.), flax (Linum usitatissumum), geraniums (Pelargonium spp.), grapefruit (Citrus x paradisi), grapes (Vitus spp.) including wine grapes (Vitus vinifera), guava (Psidium guajava), irises (Iris spp.), lemon (Citrus limon), lettuce (Lactuca sativa), limes (Citrus spp.), maize (Zea mays), mango (Mangifera indica), mangos
  • the plant cell is a rice cell (Oryza sativa), a soybean cell (Glycine max), tobacco (Nicotiana tabacum), tomato (Lycopersicon esculentum), or maize (Zea mays).
  • the plant cell (including protoplasts) is an isolated plant cell or part of an isolated plant tissue (e.g., tissue culture).
  • the isolated plant cell is obtained or isolated from a whole plant or a plant part or tissue, for example (and without limitation), an intact nodal bud, a shoot apex or shoot apical meristem, a root apex or root apical meristem, lateral meristem, intercalary meristem, a seedling (e.g., a germinating seed or small seedling or a larger seedling with one or more true leaves), a whole seed (e.g., an intact seed, or a seed with part or all of its seed coat removed or treated to make permeable), a halved seed or other seed fragment, an embryo 9582-111817-02 (e.g., a mature dissected zygotic embryo, a developing embryo, a dry or rehydrated or freshly excised embryo), or callus.
  • a seedling e.g., a germinating seed or small seedling or a larger seedling with one or more true leaves
  • the isolated plant cell is a protoplast.
  • transformed plant cells including protoplasts
  • a transformed plant cell is regenerated to a whole plant.
  • the methods further include one or more steps of growing or regenerating a plant from a transformed plant cell, for example, a transformed plant cell including, producing or expressing a nucleic acid disclosed herein (e.g., a nucleic acid encoding a Cas protein disclosed herein), or including an induced genetic modification or altered gene expression as disclosed herein, thereby generating a modified plant.
  • the grown or regenerated plant contains at least some cells or tissues producing or expressing the transformed nucleic acid or including the induced genetic modification.
  • a callus is produced from a transformed plant cell, and plantlets and plants are produced from the callus.
  • whole seedlings or plants are grown directly from a transformed plant cell without a callus stage.
  • whole seedlings and plants are grown or regenerated from transformed plant cells produced by the methods disclosed herein. Encompassed by this disclosure is the seed of such plants.
  • the grown or regenerated plant exhibits a phenotype associated with expression of the nucleic acid, the induced genetic modification, or altered gene expression.
  • Non-limiting phenotypes include herbicide resistance, improved tolerance of abiotic stress (e.g., tolerance of temperature extremes, drought, or salt) or biotic stress (e.
  • resistance to bacterial or fungal pathogens improved utilization of nutrients or water, modified lipid, carbohydrate, or protein composition, improved flavor or appearance, improved storage characteristics (e.g., resistance to bruising, browning, or softening), increased yield, altered morphology (e.g., floral architecture or color, plant height, branching, root structure), or expression of a selectable marker.
  • the methods can include a selection step of selecting transformed plant cells (or seedlings or plants grown or regenerated therefrom) with a desired phenotype, for example, transformed plant cells (or seedlings or plants) can be exposed to conditions permitting expression of a phenotype of interest; e.g., selection for herbicide resistance can include exposing the population of plant cells (or seedlings or plants) to an amount of herbicide or other substance that inhibits growth or is toxic, allowing identification and selection of those resistant plant cells (or seedlings or plants) that survive treatment. Plant cells (or seedlings or plants grown or regenerated therefrom) can be selected based on manifestation of a desired phenotype. Such plants can be selected, for example, for further analysis or plant breeding.
  • the plant cell can be haploid, diploid, or polyploid. In some aspects, the plant cell is haploid or can be induced to become haploid. Examples of haploid cells include but are not limited to plant cells obtained from haploid plants and plant cells obtained from reproductive tissues, e.g., from flowers, developing flowers or flower buds, ovaries, ovules, megaspores, anthers, pollen, and microspores.
  • the method of modifying the genetic 9582-111817-02 material of the plant cell can further include a step of chromosome doubling (e.g., by spontaneous chromosomal doubling by meiotic non-reduction, or by using a chromosome doubling agent such as colchicine, oryzalin, or trifluralin) to produce a doubled haploid plant cell that is homozygous for the induced genetic modification.
  • chromosome doubling e.g., by spontaneous chromosomal doubling by meiotic non-reduction, or by using a chromosome doubling agent such as colchicine, oryzalin, or trifluralin
  • aspects of the disclosure are related to haploid plant cells having the altered target nucleotide sequence as well as a doubled haploid plant cells or a doubled haploid plant that is homozygous for an induced genetic modification.
  • Another aspect of the disclosure is related to a hybrid plant having at least one parent plant that is a doubled haploid plant provided by the method.
  • Production of doubled haploid plants by these methods provides homozygosity in one generation, instead of requiring several generations of self-crossing to obtain homozygous plants; this may be particularly advantageous in slow-growing plants, such as fruit and other trees, or for producing hybrid plants that are offspring of at least one doubled-haploid plant.
  • a class 1 type I CRISPR-Cas system comprising: a CRISPR-associated complex for antiviral defense (CASCADE) complex comprising a plurality of Cas proteins and an artificial guide RNA (gRNA), wherein the CASCADE complex is: i) a Methylomonas methanica type IF system CASCADE complex; ii) a Sphingobium amiense type IC system CASCADE complex; iii) an Alteromonas mediterranea type IFv system CASCADE complex; or iv) a Pragia fontium type IF system CASCADE complex.
  • CASCADE complex is: i) a Methylomonas methanica type IF system CASCADE complex; ii) a Sphingobium amiense type IC system CASCADE complex; iii) an Alteromonas mediterranea type IFv system CASCADE complex; or iv) a Pragia font
  • the class 1 type I CRISPR-Cas system of any one of the prior clauses comprising: i) the Methylomonas methanica type IF system CASCADE complex, wherein the plurality of Cas proteins comprises or consists of the Methylomonas methanica Cas5f, Cas6, Cas7f, and Cas8f; ii) the Sphingobium amiense type IC system CASCADE complex, wherein the plurality of Cas proteins comprises or consists of the Sphingobium amiense Cas5, Cas7, Cas8c, and Cas11; iii) the Alteromonas mediterranea type IFv system CASCADE complex, wherein the plurality of Cas proteins comprises or consists of the Alteromonas mediterranea Cas5fv, Cas6, and Cas7fv; or 9582-111817-02 iv) the Pragia fontium type IF system CASCADE complex,
  • the class 1 type I CRISPR-Cas system of any one of the prior clauses further comprising a Cas3, Cas2-Cas3, or a Fok1 endonuclease domain.
  • Clause 5. The class 1 type I CRISPR-Cas system of any one of the prior clauses, comprising: i) the Methylomonas methanica type IF system CASCADE complex, wherein: a) the Cas5f comprises at least 95% sequence identity to SEQ ID NO: 1; b) the Cas6 comprises at least 95% sequence identity to SEQ ID NO: 2; c) the Cas7f comprises at least 95% sequence identity to SEQ ID NO: 3; d) the Cas8f comprises at least 95% sequence identity to SEQ ID NO: 4; and/or e) the Cas2-Cas3 comprises at least 95% sequence identity to SEQ ID NO: 5; ii) the Sphingobium amiense type IC system CASCADE complex, wherein:
  • the class 1 type I CRISPR-Cas system of any one of the prior clauses comprising: i) the Methylomonas methanica type IF system CASCADE complex, wherein: a) the Cas5f comprises or consists of SEQ ID NO: 1; b) the Cas6 comprises or consists of SEQ ID NO: 2; c) the Cas7f comprises or consists of SEQ ID NO: 3; d) the Cas8f comprises or consists of SEQ ID NO: 4; and/or e) the Cas2-Cas3 comprises or consists of SEQ ID NO: 5; ii) the Sphingobium amiense type IC system CASCADE complex, wherein: a) the Cas5 comprises or consists of SEQ ID NO: 6; 9582-111817-02 b) the Cas7 comprises or consists of SEQ ID NO: 7; c) the Cas8c comprises or consists of SEQ ID NO: 8; d) the Cas5f
  • the class 1 type I CRISPR-Cas system of any one of the prior clauses comprising: i) the Methylomonas methanica type IF system CASCADE complex, wherein: a) the Cas5f comprises or consists of SEQ ID NO: 1; b) the Cas6 comprises or consists of SEQ ID NO: 2; c) the Cas7f comprises or consists of SEQ ID NO: 3; d) the Cas8f comprises or consists of SEQ ID NO: 4; and e) the Cas2-Cas3 comprises or consists of SEQ ID NO: 5; ii) the Sphingobium amiense type IC system CASCADE complex, wherein: a) the Cas5 comprises or consists of SEQ ID NO: 6; b) the Cas7 comprises or consists of SEQ ID NO: 7; c) the Cas8c comprises or consists of SEQ ID NO: 8; d) the Cas11 comprises or consists of SEQ
  • Clause 8 The class 1 type I CRISPR-Cas system of any one of the prior clauses, wherein the gRNA is a dead guide RNA (dgRNA).
  • dgRNA dead guide RNA
  • Clause 9 The class 1 type I CRISPR-Cas system of any one of the prior clauses, further comprising an effector molecule linked to at least one of the plurality of Cas proteins, wherein the effector molecule is a transcriptional activator, a transcriptional repressor, a base editor, a reporter, a nuclear localization signal (NLS), or a combination of two or more thereof.
  • a recombinant nucleic acid molecule encoding at least one of the plurality of Cas proteins of any one of the prior clauses. Clause 11.
  • the recombinant nucleic acid molecule of clause 10 encoding: i) the Methylomonas methanica type IF system CASCADE complex; ii) the Sphingobium amiense type IC system CASCADE complex; iii) the Alteromonas mediterranea type IFv system CASCADE complex; or iv) the Pragia fontium type IF system CASCADE complex.
  • a recombinant nucleic acid molecule encoding one or more Cas proteins of a Type 1 CRISPR system wherein the one or more Cas proteins are from: i) a Methylomonas methanica type IF system; ii) a Sphingobium amiense type IC system; iii) an Alteromonas mediterranea type IFv system; or iv) a Pragia fontium type IF system.
  • the recombinant nucleic acid molecule of clause 13, comprising: i) a nucleic acid molecule encoding the one or more Cas proteins from the Methylomonas methanica type IF system, wherein the Cas proteins comprise a Methylomonas methanica Cas2-Cas3, Cas5f, Cas6, Cas7f, and/or Cas8f; ii) a nucleic acid molecule encoding the one or more Cas proteins from the Sphingobium amiense type IC system, wherein the one or more Cas proteins comprise a Sphingobium amiense Cas3, Cas5, Cas7, Cas8c, and/or Cas11; iii) a nucleic acid molecule encoding the one or more Cas proteins from the Alteromonas mediterranea type IFv system, wherein the one or more Cas proteins comprise an Alteromonas mediterranea Cas2-Cas
  • Clause 16 The recombinant nucleic acid molecule of any one of clauses 13 to 15, comprising: i) the nucleic acid molecule encoding the one or more Cas proteins from the Methylomonas methanica type IF system, wherein: a) the Cas5f comprises at least 95% sequence identity to SEQ ID NO: 1; b) the Cas6 comprises at least 95% sequence identity to SEQ ID NO: 2; c) the Cas7f comprises at least 95% sequence identity to SEQ ID NO: 3; d) the Cas8f comprises at least 95% sequence identity to SEQ ID NO: 4; and/or e) the Cas2-Cas3 comprises at least 95% sequence identity to SEQ ID NO: 5; ii) the nucleic acid molecule encoding the one or more Cas proteins from the Sphingobium amiense type IC system, wherein: a) the Cas5 comprises at least 95% sequence identity to SEQ ID NO: 6; b) the Ca
  • Clause 18 The recombinant nucleic acid molecule of any one of clauses 13 to 17, comprising: i) the Methylomonas methanica type IF system, wherein the nucleic acid molecule comprises one or more of SEQ ID NOs: 20-24, or a degenerate variant thereof; ii) the Sphingobium amiense type IC system, wherein the nucleic acid molecule comprises one or more of SEQ ID NOs: 25-29, or a degenerate variant thereof; 9582-111817-02 iii) the Alteromonas mediterranea type IFv system, wherein the nucleic acid molecule comprises one or more of SEQ ID NOs: 30-33, or a degenerate variant thereof; or iv) the Pragia fontium type IF system, wherein the nucleic acid molecule comprises one or more of SEQ ID NOs: 34-38, or a degenerate variant thereof.
  • nucleic acid molecule of any one of clauses 13 to 18, comprising: i) the Methylomonas methanica type IF system, wherein the nucleic acid molecule comprises each of SEQ ID NOs: 20-23, or degenerate variants thereof; ii) the Sphingobium amiense type IC system, wherein the nucleic acid molecule comprises each of SEQ ID NOs: 25-28, or degenerate variants thereof; iii) the Alteromonas mediterranea type IFv system, wherein the nucleic acid molecule comprises each of SEQ ID NOs: 30-32, or degenerate variants thereof; or iv) the Pragia fontium type IF system, wherein the nucleic acid molecule comprises each of SEQ ID NOs: 34-37, or degenerate variants thereof.
  • Clause 20 The recombinant nucleic acid molecule of any one of clauses 10 to 19, wherein the nucleic acid molecule is codon optimized for expression in plants.
  • Clause 21 A vector comprising the recombinant nucleic acid molecule of any one of clauses 10 to 20.
  • Clause 22. A host cell comprising the class 1 type I CRISPR-Cas system of any one of clauses 1 to 9, the recombinant nucleic acid of any one of clauses 10 to 20, or the vector of clause 21.
  • a method of modifying a target nucleic acid sequence or altering expression of a target nucleic acid in a cell comprising: introducing the class 1 type I CRISPR-Cas system of any one of clauses 1 to 9 into the cell.
  • Clause 24 The method of clause 23, wherein introducing comprises transforming the cell with one or more nucleic acid molecules or vectors encoding the class 1 type I CRISPR-Cas system of any one of clauses 1 to 9, and/or transforming the cell with a ribonucleoprotein (RNP) particle comprising the class 1 type I CRISPR-Cas system of any one of clauses 1 to 9.
  • RNP ribonucleoprotein
  • Clause 32 A plant generated from the cell or tissue culture of any one of clauses 28 to 30. 9582-111817-02 Clause 33. Seed of the plant of clause 32.
  • EXAMPLES The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified.
  • Maize codon optimized Cas genes were ordered (using IDT codon optimization tool) as synthetic DNA and cloned into the modified Golden Gate and Gateway LR assembly cloning vectors to ensure expression from a single T-DNA vector (Lowder et al., Plant Physiol 169:971-985, 2015) encoding CASCADE, Cas3, and either mature gRNA or gRNA to be self-processed (FIGS.5A-5C).
  • Example 2 Characterization of top CRIPSR editing candidates
  • the Type I systems were tested using an in vitro transcription/translation assay (IVTT; Arbor Biosciences) based on GPF plasmid restriction measured as a decrease in the rate/final level of fluorescence in the sample containing type I systems relative to the negative control containing a rice gene targeting guide with no homology to the reporter GFP plasmid.
  • the IVTT assay was performed as described in Wimmer et al.2022 (“Rapid cell-free characterization of multi-subunit CRISPR effectors and transposons.” Molecular Cell, 82(6): 1210-1224).
  • the IVTT assay showed a reduction in GFP fluorescence, indicating reduction in GFP transcription through binding or cleavage of GFP.
  • Type I systems achieved specific DNA targeting.
  • Type I systems were also tested in rice protoplasts. Control of transformation efficiency was above 90% as determined by fluorescence of the protoplasts transformed with GFP expressing T- DNA vector.
  • Genome editing control represented a CRIPSR-Cas12a system editing previously tested target site. All tested novel Type I systems were screened for activity in at least 5 biological replicates. After the assay was completed, DNA was isolated and PCR amplified using Phire Plant Direct PCR Kit (Thermo Fisher, cat. #: F130WH) to produce PCR amplicons of up to 5 kbp in size with the Type I restriction site located in the middle of the amplicon (FIG.6).
  • PCR amplicons were purified, desalinated and nanopore sequenced to determine novel type I system’s activity.
  • Methylomonas methanica MC09 – TIF demonstrated in two biological replicates approximately 1.5 kbp unidirectional deletion upstream from the OsGS3-gR1 target site (FIGS.7-8).
  • a list of CRISPR type IC and IF systems was curated.
  • Results from an IVTT assay demonstrated prevention of GFP transcription, indicating successful DNA editing.
  • results from rice protoplast assay of screening of novel Type I systems demonstrated unidirectional cleaving activity as detected by Nanopore sequencing.
  • CRISPR Type IC and IF systems are functional, and useful for a variety of applications including genome, epigenome editing, and transcriptional regulation.
  • 9582-111817-02 Example 3 Stable Transformation of Methylomonas methanica MC09 – TIF in Rice
  • Type IF MM Metalomonas methanica MC09
  • Two constructs were selected for Agrobacterium-mediated stable transformation in rice cv. Kitaake.
  • Genomic DNA was extracted from T0 plants and long-range amplicons were amplified from plants targeting OsGS3 and OsGW2, respectively. Nested PCR revealed the presence of large deletions (FIG.9).
  • PCR products were cleaned up using a Qiagen PCR purification kit and subjected to Oxford Nanopore Technologies® DNA nanopore sequencing.
  • Raw reads were processed and mapped to a reference sequence and sequencing coverage was viewed using Integrated Genomics Viewer (IGV) (FIG.10A).
  • IGF Integrated Genomics Viewer
  • FIG.10B Genome editing summary statistics are shown in FIG.10B. Regions of low coverage revealed large, bidirectional deletions generated by CRISPR Type IF MM at both target sites (see, FIGS.11A-11D). These results suggest that this Type IF MM system is functional in rice plants and can introduce deletions spanning kilobases in size, with some plants exhibiting deletions greater than 3.5 kilobases.
  • a Fok1 nuclease domain e.g., SEQ ID NO: 102
  • a peptide linker e.g., a glycine-serine linker
  • a pair of artificial gRNAs are designed to target the same locus in a PAM-out orientation, with a 15 to 20bp gap in between the two target sites.

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Abstract

La présente invention concerne des systèmes CRISPR-Cas de type I de classe 1, en particulier des systèmes provenant de Methylomonas methanica, Sphingobium amiense, Alteromonas mediterranea et Pragia fontium. Sont également divulgués des molécules d'acide nucléique recombinant et des vecteurs codant un ou plusieurs composants des systèmes CRISPR-Cas de type I de classe 1 divulgués. L'invention concerne, en outre, des procédés de modification d'une séquence d'acide nucléique cible ou de modification de l'expression d'un acide nucléique cible dans une cellule, par exemple, dans une cellule végétale.
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Citations (3)

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Publication number Priority date Publication date Assignee Title
WO2019241452A1 (fr) * 2018-06-13 2019-12-19 Caribou Biosciences, Inc. Composants en cascade modifiés et complexes en cascade
US11439712B2 (en) * 2014-04-08 2022-09-13 North Carolina State University Methods and compositions for RNA-directed repression of transcription using CRISPR-associated genes
US20230056577A1 (en) * 2018-12-17 2023-02-23 The Broad Institute, Inc. Crispr-associated transposase systems and methods of use thereof

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Publication number Priority date Publication date Assignee Title
US11439712B2 (en) * 2014-04-08 2022-09-13 North Carolina State University Methods and compositions for RNA-directed repression of transcription using CRISPR-associated genes
WO2019241452A1 (fr) * 2018-06-13 2019-12-19 Caribou Biosciences, Inc. Composants en cascade modifiés et complexes en cascade
US20230056577A1 (en) * 2018-12-17 2023-02-23 The Broad Institute, Inc. Crispr-associated transposase systems and methods of use thereof

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BODEN RICH, CUNLIFFE MICHAEL, SCANLAN JULIE, MOUSSARD HÉLÈNE, KITS K. DIMITRI, KLOTZ MARTIN G., JETTEN MIKE S. M., VUILLEUMIER STÉ: "Complete Genome Sequence of the Aerobic Marine Methanotroph Methylomonas methanica MC09", JOURNAL OF BACTERIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 193, no. 24, 15 December 2011 (2011-12-15), US , pages 7001 - 7002, XP093393498, ISSN: 0021-9193, DOI: 10.1128/jb.06267-11 *

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