WO2019083532A1 - Identification de protéines d'édition génique et d'excision génique (de type cas9 et argonaute) à partir d'autres organismes - Google Patents

Identification de protéines d'édition génique et d'excision génique (de type cas9 et argonaute) à partir d'autres organismes

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Publication number
WO2019083532A1
WO2019083532A1 PCT/US2017/058397 US2017058397W WO2019083532A1 WO 2019083532 A1 WO2019083532 A1 WO 2019083532A1 US 2017058397 W US2017058397 W US 2017058397W WO 2019083532 A1 WO2019083532 A1 WO 2019083532A1
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WIPO (PCT)
Prior art keywords
chlorella
nuclease
streptococcus
var
atcc
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PCT/US2017/058397
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English (en)
Inventor
Thomas MALCOLM
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Excision BioTherapeutics Inc
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Excision BioTherapeutics Inc
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Priority to PCT/US2017/058397 priority Critical patent/WO2019083532A1/fr
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Anticipated expiration legal-status Critical
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    • 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
    • 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

Definitions

  • the present invention relates to methods of identifying gene editing and gene excising proteins. More specifically, the present invention relates to methods of identifying alternative Cas9-like and Argonaute-like gene editors and gene excisors from microbial sources.
  • nucleases Gene editing allows DNA or RNA to be inserted, deleted, or replaced in an organism's genome by the use of nucleases.
  • nucleases There are several types of nucleases currently used, including meganucleases, zinc finger nucleases, transcription activatorlike effector-based nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas nucleases. These nucleases can create site- specific double strand breaks of the DNA in order to edit the DNA.
  • Meganucleases have very long recognition sequences and are very specific to DNA. While meganucleases are less toxic than other gene editors, they are expensive to construct, as not many are known and mutagenesis must be used to create variants that recognize specific sequences.
  • Both zinc-finger and TALEN nucleases are non-specific for DNA but can be linked to DNA sequence recognizing peptides. However, each of these nucleases can produce off-target effects and cytotoxicity, and require time to create the DNA sequence recognizing peptides.
  • CRISPR-Cas nucleases are derived from prokaryotic systems and can use either the Cas9 nuclease or the Cpf1 nuclease for DNA editing.
  • CRISPR is an adaptive immune system found in many microbial organisms. While the CRISPR system was not well understood, it was found that there were genes associated to the CRISPR regions that coded for exonucleases and/or helicases, called CRISPR-associated proteins (Cas).
  • CRISPR-associated proteins Cas.
  • Several different types of Cas proteins were found, some using multi- protein complexes (Type I), some using singe effector proteins with a universal tracrRNA and crRNA specific for a target DNA sequence (Type II), and some found in archea (Type III).
  • Cas9 (a Type II Cas protein) was discovered when the bacteria Streptococcus thermophilus was being studied and an unusual CRISPR locus was found (Bolotin, et al. 2005). It was also found that the spacers share a common sequence at one end (the protospacer adjacent motif PAM), and is used for target sequence recognition. Cas9 was not found with a screen but by examining a specific bacteria. CRISPR can also use C2c2 nuclease for targeting RNA. Small guide RNAs are used with the nucleases to recognize DNA or RNA.
  • Argonaute proteins are proteins of the PIWI protein superfamily that contain a PIWI (P element-induced wimpy testis) domain, a MID (middle) domain, a PAZ (Piwi- Argonaute-Zwille) domain and an N-terminal domain.
  • Argonaute proteins are capable of binding small RNAs, such as microRNAs, small interfering RNAs (siRNAs), and Piwi- interacting RNAs.
  • Argonaute proteins can be guided to target sequences with these RNAs in order to cleave imRNA, inhibit translation, or induce imRNA degradation in the target sequence.
  • Argonaute proteins There are several different human Argonaute proteins, including AGO1 , AGO2, AGO3, and AGO4 that associate with small RNAs.
  • AGO2 has slicer ability, i.e. acts as an endonuclease.
  • Argonaute proteins can be used for gene editing. Endonucleases from the Argonaute protein family (from Natro no bacterium gregoryi Argonaute) also use oligonucleotides as guides to degrade invasive genomes. Work by Gao et al has shown that the Natro no bacterium gregoryi Argonaute (NgAgo) is a DNA- guided endonuclease suitable for genome editing in human cells.
  • NgAgo Natro no bacterium gregoryi Argonaute
  • NgAgo binds 5' phosphorylatedsingle-stranded guide DNA (gDNA) of -24 nucleotides, efficiently creates site-specific DNA double-strand breaks when loaded with the gDNA.
  • gDNA 5' phosphorylatedsingle-stranded guide DNA
  • the NgAgo-gDNA system does not require a protospacer-adjacent motif (PAM), as does Cas9, and preliminary characterization suggests a low tolerance to guide-target mismatches and high efficiency in editing (G+C)-rich genomic targets.
  • PAM protospacer-adjacent motif
  • the present invention provides for a method of identifying a gene editor and gene excisor nuclease (Cas9-like and Argonaute-like) protein with an assay, by stressing a microbial strain with bacteriophage infection, separating nuclease proteins from non-nuclease proteins, and identifying the nuclease proteins.
  • the present invention also provides for a method of identifying Cas9 nuclease activity in a nuclease protein, by verifying sgRNA (and/or sgDNA) and nuclease activity of a nuclease protein, and determining a PAM sequence for the nuclease protein.
  • the present invention also provides for a method of identifying Argonaute nuclease activity in a nuclease protein, by verifying sgRNA (and/or sgDNA) and nuclease activity of a nuclease protein.
  • the present invention also provides for any molecule with gene editing and/or gene excising capabilities obtained and isolated by the methods described above.
  • the present invention provides for a gene excisor obtained and isolated by the methods described above.
  • the present invention is generally directed to methods of identifying gene editors and gene excisors with a biochemical and function-based screen, as well as gene editors and gene excisors obtained and isolated by the methods described herein.
  • test refers to a procedure that determines the amount or presence or activity of a particular constituent of a mixture or sample.
  • gene editor refers to a molecule that is able to target DNA or RNA and make additions, deletions, mutations, and preferably excisions of entire genes.
  • gene excisor refers to one or more molecules that are able to target DNA or RNA and excise and delete regions of entire genes or gene clusters, in any targeting combination (differing gRNAs or gDNAs) thereof and in any combination of gene excisor, i.e. the use of different gene editors/excisors in combination with one another.
  • Cas9-like refers to a gene editor or gene excisor that has the ability to act like Cas9, i.e. acts as an RNA-guided DNA endonuclease enzyme that is able to cleave any sequence complementary to guide RNA.
  • Argonaute-like refers to a gene editor or gene excisor that has the ability to act like Argonaute, i.e. acts as a RNA-guided or DNA-guided endonuclease enzyme that is able to cleave any sequence complementary to guide RNA or guide DNA.
  • Nuclease refers to an enzyme that is able to cleave the phosphodiester bonds between nucleotide subunits of nucleic acids.
  • sgRNA refers to single guide RNA.
  • sgDNA refers to single guide DNA.
  • the present invention provides for a method of identifying a gene editor and gene excisor nuclease (Cas9-like and Argonaute-like) protein with an assay, by stressing a microbial strain with bacteriophage infection, separating nuclease proteins from non-nuclease proteins, and identifying the nuclease proteins.
  • This method can be used to find new gene editor and gene excisor nuclease proteins. Each of these steps are further described in Example 1 . While it is envisioned that the gene editor nuclease is a protein, this method can also identify any type of molecule that has gene editing and gene excising capabilities.
  • the gene editor nuclease protein identified is a gene excisor, meaning a molecule that is capable of excising an entire gene or target sequence in a genome instead of merely altering it.
  • the microbial strain can be any suitable bacteria, such as, but not limited to, Acetobacter aurantius, Acinetobacter bitumen, Actinomyces israelii, Agrobacterium radiobacter, Agrobacterium tumefaciens, Anaplasma, Anaplasma phagocytophilum, Azorhizobium caulinodans, Azotobacter vinelandii, viridans streptococci, keshav, Bacillus, Bacillus anthracis, Bacillus brevis, Bacillus cereus, Bacillus fusiformis, Bacillus licheniformis, Bacillus megaterium, Bacillus mycoides, Bacillus stearothermophilus, Bacillus subtilis, Bacteroides, Bacteroides fragilis,
  • the bacteria is any of Pyrinomonas methylaliphatogenes type strain K22T, Thermodesulfobacterium geofontis (ATCC BAA 2454), Thermus thermophilus (ATCC 27634), Haloarcula marismortui (ATCC 43049), Moritella marina MP-1 (ATCC 15381 ), Methylacidiphilum infernorum V4, Polaromonas vacuolata (ATCC 51984), Halobacterium salinarum (ATCC 33170), Methanococcus jannaschii (ATCC 99631 ), Thermus aquaticus (ATCC 25104), Sulfolobus acidocaldarius (ATCC 33909), Methanopyrus kandleri (ATCC BAA 1075), Natro no bacterium gregoryi (ATCC 43098), Haloferax volcanii (ATCC 29605), Staphylococcus aureus
  • the microbial strain can also be any suitable yeast, blue green algae, or chlamydomonas.
  • the yeast can be, but is not limited to, Cryptococcus curvatus, Cryptococcus terricolus, Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis, Candida 107, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces bayanus, Saccharomyces cerevisiae, any Cryptococcus, C. neoformans, C. bogoriensis, Yarrowia lipolytica, Apiotrichum curvatum, T. bombicola, T. apicola, T. petrophilum, C. tropicalis, C. lipolytica, or Candida albicans.
  • the blue green algae can be, but is not limited to, Achnanthes orientalis, Agmenellum spp., Amphiprora hyaline, Amphora coffeiformis, Amphora coffeiformis var. linea, Amphora coffeiformis var. punctata, Amphora coffeiformis var. taylori, Amphora coffeiformis var. tenuis, Amphora americanissima, Amphora strigissima var. .
  • Chaetoceros sp. Chlamydomas perigranulata, Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulate, Chlorella desiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. actophila, Chlorella infusionum var.
  • Chlorella kessleri Chlorella lobophora
  • Chlorella luteoviridis Chlorella luteoviridis var. aureoviridis
  • Chlorella luteoviridis var. lutescens Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella ovalis, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella protothecoides var. acidicola, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var.
  • the clamydomonas can be, but is not limited to, Chlamydomonas reinhardtii, Chlamydomonas caudate Wille, Chlamydomonas moewusii, Chlamydomonas nivalis, or Chlamydomonas ovoidae.
  • the bacteriophage used can be any suitable bacteriophage, such as, but not limited to, those from the myoviridae (T4 phage, Mu, PBSX, P1 Puna-like, P2, I3, Beep 1 , Beep 43, Beep 78), siphoviridae (gamma phage, T5 phage, phi, C2, L5, HK97, N15), podoviridae (T7 phage, T3 phage, phi29, P22, P37), lipothrixviridae (acidianus filamentous virus 1 ), rudiviridae (sulfolobus islandicus rod-shaped virus 1 ), ampullaviridae, bicaudaviridae, clavaviridae, corticoviridae, cystoviridae, fuselloviridae, globuloviridae, guttaviridae, inoviridae (M13), leviviridae (MS
  • nuclease proteins can be accomplished by flowing the stressed microbial strain through a beaded column, or by any other suitable separation method.
  • the nuclease proteins can associate with sgRNA (or sgDNA) in the column, while the non-nuclease proteins and any other cell products can flow through.
  • the nuclease proteins can then be identified by any method known in the art, such as mass spectrometry.
  • the present invention also provides for a method of identifying Cas9 nuclease activity in a nuclease protein, by verifying sgRNA and nuclease activity of a nuclease protein (preferably identified by the above method), and determining a PAM sequence for the nuclease protein.
  • This method can further include the steps of validating Cas9 nuclease activity by applying the nuclease protein to a yeast in vivo system and detecting a colored yeast colony. Each of these steps are further described in Example 1 below. Both verifying sgRNA and nuclease activity and determining the PAM sequence can be accomplished by using fluorescence assays as described below.
  • the present invention also provides for a method of identifying Argonaute nuclease activity in a nuclease protein, by verifying sgRNA or sgDNA and nuclease activity of a nuclease protein (preferably identified by the above method).
  • this method can include determining a PAM sequence (if necessary) for the nuclease protein, as Argonautes do not necessarily have a PAM sequence.
  • This method can further include the steps of validating Argonaute nuclease activity by applying the nuclease protein to a yeast in vivo system and detecting a colored yeast colony. Each of these steps are further described in Example 1 below. Both verifying sgRNA or sgDNA and nuclease activity and determining the PAM sequence (if necessary) can be accomplished by using fluorescence assays as described below.
  • This assay can be used for other applications.
  • the assay can be used to screen for inhibitors of a specific Cas or Argonaute protein.
  • mutations in Cas9-like and Argonaute-like proteins can be made to screen for changes in the PAM sequence (if necessary for Argonaute-like nucleases).
  • purified protein can be submitted for crystallization studies.
  • the present invention also provides for any molecule (and in particular nuclease proteins) with gene editing and/or gene excising capabilities obtained and isolated by the methods described above, and particularly for therapeutic use in mammalian cells.
  • Gene editing and/or gene excising capabilities include the ability to edit RNA or DNA in a genome (addition, deletion, mutation, or entire excision of target genetic material) and preferably with the use of two or more guide RNAs (gRNAs) or guide DNAs (gDNAs).
  • gRNAs guide RNAs
  • gDNAs guide DNAs
  • the molecules identified can be used in many different treatments for humans and animals, such as for virus infection. More preferably, the present invention provides for a gene excisor obtained and isolated by the methods described above for therapeutic use in mammalian cells.
  • the gene excisor is a molecule that has the ability to excise entire genes or target sequences in a genome of an organism.
  • the molecule or gene excisor can be isolated from any of the microbial strains as described above, but can preferably be isolated from a bacteria of Pyrinomonas methylaliphatogenes type strain K22T, Thermodesulfobacterium geofontis (ATCC BAA 2454), Thermus thermophilus (ATCC 27634), Haloarcula marismortui (ATCC 43049), Moritella marina MP-1 (ATCC 15381 ), Methylacidiphilum infernorum V4, Polaromonas vacuolata (ATCC 51984), Halobacterium salinarum (ATCC 33170), Methanococcus jannaschii (ATCC 99631 ), Thermus aquaticus (ATCC 25104), Sulfolobus acidocaldarius (ATCC 33909), Me
  • Microbial strains can be purchased from ATCC (commercially available), grown under the proper conditions, stressed with bacteriophage infection and processed to produce whole cell extracts. Bacteriophage infection has been demonstrated to increase the expression of CRISPR regions, along with the linked Editor, within bacterial genomes.
  • the whole cell extracts can be pre-cleared for non- guide RNA (or non-guided DNA) and non-specific bead binding by incubation with total cellular RNA conjugated to acrylamide beads.
  • the flowthrough from this column can be incubated with magnetic beads prepared from biotinylated (single-type) gRNA (or single type gDNA) bound to the streptavidin-linked magnetic beads.
  • the length of the streptavidin-linker moiety can be optimized to ensure the sgRNA (or sgDNA) assumes the proper 3-dimensional structure in relation (but not limited to) to the structure of cas9-like or Argonaute-like nucleases.
  • the notion is that the putative Cas9-like or Argonaute-like molecules will associate with the sgRNAs (or sgDNAs and sgRNAs in the case of Argonaute-like nucleases) and those proteins that do not associate will flowthrough the column.
  • the column can be washed with the same buffer as was used to make the whole cell extracts. Bound proteins can be eluted with the buffer plus urea and SDS for collection.
  • the samples can be separated by SDS-PAGE (8 percent, but not limited to, as all the editors so far have been approximately 100KDa).
  • the gel(s) can be silver stained and the bands (those bands not also present in the scrambled sgRNA (or sgDNAs and sgRNAs in the case of Argonaute-like nucleases) column) can be excised and identified using Mass Spectrometry (commercially available).
  • Mass Spectrometry commercially available.
  • the identified proteins can be prioritized based on a) presence of a nuclease domain, b) expressibility, c) crystal structure, d) structural information not obtained by crystallization, e) annotation(s), f) known RNA and DNA -associated protein and g) size. Based on the priority list, proteins can be cloned using PCR from DNA of the organism from which they were originally isolated. A pilot assay can be performed with extracts from S. aureus to optimize the assay conditions and steps and to demonstrate the isolation and purification efficiency.
  • a pilot assay can be performed with the S. aureus Cas9 or the N.gregoryi Argonaute to optimize the assay conditions and steps and to demonstrate the assay's utility.
  • the source of the Cas or Argonaute protein will be from bacterial expression, tagging the protein with a HIS10 tag for easy purification. If there is a problem with expression in bacteria, then S. cerevisiae can be used as the expression host.
  • the target dsDNA can be synthesized by hybridizing defined complementary oligos containing a) the guide recognition sequence, b) a PAM sequence and c) a fluorescent tag at the 5' end for the sense strand and a quencher tag at the 5' end of the non-sense strand.
  • the proximity of the fluorophore to the quencher will quench/mask the fluorescence signal.
  • the Cas9-like (or Argonaute-like nuclease if applicable) molecule catalyzes the double strand cleavage of the target dsDNA, the fluorophore becomes separated from the quencher, allowing for an increase in fluorescence intensity, which can be measured either as an end point assay or as a function of time.
  • a standard curve of fluorescence intensity using only the sense strand can be used to determine the amount of cleaved target dsDNA, and therefore, the efficiency of cleavage.
  • oligodeoxynucleotide libraries can be set up in a number of 96-well microtiter plates and frozen so that when a Cas9-like (or Argonaute-like nuclease if applicable) nuclease needs to be examined, plates can just be thawed and the Cas (or Argonaute if applicable) protein added.
  • Validation can be performed using a yeast in vivo system.
  • a yeast in vivo system There are systems published where a positive result produces a colored yeast colony (the system uses mutation in the ergosterol pathway to build up beta-carotine, which turns the yeast colonies orange).

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Abstract

L'invention concerne un procédé d'identification d'une protéine de nucléase d'édition génique et d'excision génique (de type Cas9 et argonaute) par un dosage, par la mise sous stress d'une souche microbienne par une infection par un bactériophage, la séparation de protéines de nucléase des protéines non nucléase et l'identification des protéines de nucléase. L'invention concerne également un procédé d'identification de l'activité de nucléase Cas9 dans une protéine de nucléase, par la vérification de l'activité d'ARNsg (et/ou d'ADNsg) et de nucléase d'une protéine de nucléase et par la détermination d'une séquence PAM pour la protéine de nucléase. L'invention concerne en outre un procédé d'identification de l'activité de nucléase argonaute dans une protéine de nucléase, par la vérification de l'activité d'ARNsg (et/ou d'ADNsg) et de nucléase d'une protéine de nucléase. L'invention porte également sur toute molécule pourvue de capacités d'édition génique et/ou d'excision génique obtenue et isolée par les procédés décrits ci-dessus. L'invention concerne également un exciseur génique obtenu et isolé par les procédés décrits ci-dessus.
PCT/US2017/058397 2017-10-26 2017-10-26 Identification de protéines d'édition génique et d'excision génique (de type cas9 et argonaute) à partir d'autres organismes Ceased WO2019083532A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11098297B2 (en) 2017-06-09 2021-08-24 Editas Medicine, Inc. Engineered Cas9 nucleases
US11447774B2 (en) * 2017-09-07 2022-09-20 The Board Of Trustees Of The Leland Stanford Junior University Nuclease systems for genetic engineering
US12286727B2 (en) 2016-12-19 2025-04-29 Editas Medicine, Inc. Assessing nuclease cleavage

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHYLINSKI ET AL.: "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems", RNA BIOL, vol. 10, no. 5, May 2013 (2013-05-01), pages 726 - 737, XP055116068, DOI: doi:10.4161/rna.24321 *
LANDER: "The Heroes of CRISPR", CELL, vol. 164, no. 1-2, 14 January 2016 (2016-01-14), pages 18 - 28, XP029385498, DOI: doi:10.1016/j.cell.2015.12.041 *
MICHLEWSKI ET AL.: "RNase-assisted RNA chromatography", RNA, vol. 16, no. 8, August 2010 (2010-08-01), pages 1673 - 1678, XP055595371, ISSN: 1355-8382, DOI: 10.1261/rna.2136010 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12286727B2 (en) 2016-12-19 2025-04-29 Editas Medicine, Inc. Assessing nuclease cleavage
US11098297B2 (en) 2017-06-09 2021-08-24 Editas Medicine, Inc. Engineered Cas9 nucleases
US12297466B2 (en) 2017-06-09 2025-05-13 Editas Medicine, Inc. Engineered Cas9 nucleases
US11447774B2 (en) * 2017-09-07 2022-09-20 The Board Of Trustees Of The Leland Stanford Junior University Nuclease systems for genetic engineering

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