WO2023165613A1 - Utilisation d'une exonucléase dans le sens 5' vers 3' dans un système d'édition génique, et système d'édition génique, et procédé d'édition génique - Google Patents

Utilisation d'une exonucléase dans le sens 5' vers 3' dans un système d'édition génique, et système d'édition génique, et procédé d'édition génique Download PDF

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Publication number
WO2023165613A1
WO2023165613A1 PCT/CN2023/079632 CN2023079632W WO2023165613A1 WO 2023165613 A1 WO2023165613 A1 WO 2023165613A1 CN 2023079632 W CN2023079632 W CN 2023079632W WO 2023165613 A1 WO2023165613 A1 WO 2023165613A1
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gene editing
exonuclease
editing system
cells
site
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PCT/CN2023/079632
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English (en)
Chinese (zh)
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李寅青
王沛喆
富晶晶
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清华大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N15/63—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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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 RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/16—Exonucleases active with either ribo- or deoxyribonucleic acids and producing 3'-phosphomonoesters (3.16)
    • C12Y301/16001—Spleen exonuclease (3.1.16.1), i.e. 5->3 exoribonuclease
    • 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 [CRISPRs]

Definitions

  • the invention relates to the field of biotechnology, in particular to a use of a 5' ⁇ 3' exonuclease in a gene editing system, a gene editing system and an editing method thereof.
  • the present invention aims to solve at least one of the technical problems existing in the prior art at least to a certain extent.
  • the present invention provides a use of a 5' ⁇ 3' exonuclease in a gene editing system, a gene editing system and an editing method thereof, by introducing the 5' ⁇ 3' exonuclease into the gene editing system , can improve homologous recombination repair (HDR) efficiency, reduce non-homologous end-joining repair (NHEJ), so as to realize precise gene editing.
  • HDR homologous recombination repair
  • NHEJ non-homologous end-joining repair
  • the inventors found through a large number of experiments that the exonucleic acid excision efficiency in the 5' ⁇ 3' direction of the nucleic acid damage site is the main factor limiting the editing efficiency in the dsDNA HDR technical route; and the non-mammalian endogenous 5' ⁇ 3 Exonucleases in the 'direction, especially those derived from the 5' ⁇ 3' direction of bacteriophage, have a wide range of highly active in vivo activities.
  • the present invention proposes the use of a 5' ⁇ 3' exonuclease in a gene editing system.
  • the 5' ⁇ 3' exonuclease is in a non-fused state with the site-specific nuclease in the gene editing system.
  • the present invention provides a gene editing system.
  • the gene editing system includes: a site-specific nuclease, a 5' ⁇ 3' exonuclease, and a donor DNA; wherein, the 5' ⁇ 3' exonuclease and the The site-specific nucleases are non-fused.
  • the present invention provides a method for gene editing of cells.
  • the method includes: introducing the above-mentioned gene editing system into cells.
  • Figure 17 is a histogram of gene editing efficiency at the DNMT1 locus of the U2OS cell line in Example 9 of the present invention.
  • Figure 18 is a histogram of gene editing efficiency at the mESC cell line EMX1 site in Example 9 of the present invention.
  • Figure 20 is a histogram of gene editing efficiency at TLR sites in Example 11 of the present invention.
  • Figure 24 is a histogram of gene editing efficiency at the VEGFA site in Example 11 of the present invention.
  • Figure 26 is a histogram of gene editing efficiency at the HEK3 locus in Example 12 of the present invention.
  • Figure 29 is a histogram of gene editing efficiency at the VEGFA site in Example 12 of the present invention.
  • Fig. 36 is a histogram of gene editing efficiency in the introduction of replacement mutations in Example 17 of the present invention.
  • Figure 38 is a diagram of the results of NPM1 double-color labeling immunofluorescence verification in Example 14 of the present invention.
  • Fig. 39 is a histogram of high-throughput screening results in Example 15 of the present invention.
  • gene encompasses not only chromosomal DNA present in the nucleus but also organelle DNA present in subcellular components of cells such as mitochondria.
  • the present invention proposes the use of a 5' ⁇ 3' exonuclease in a gene editing system.
  • the 5' ⁇ 3' exonuclease is in a non-fused state with the site-specific nuclease in the gene editing system.
  • the inventors improved the competitiveness of HDR pathway repair relative to NHEJ repair by adding 5' ⁇ 3' exonuclease to the gene editing system.
  • HDR pathway repair also occurs in other periods, thereby improving the efficiency of HDR pathway repair, while greatly inhibiting NHEJ; and, the present invention also found that through the addition of 5' ⁇ 3' exonuclease, it can be achieved in non-dividing cells HDR pathway.
  • 5' ⁇ 3' exonuclease in gene editing system is the use of 5' ⁇ 3' exonuclease in gene editing.
  • it refers to a A method for gene editing, the method comprising providing a 5' ⁇ 3' exonuclease, and using the 5' ⁇ 3' exonuclease for gene editing.
  • the 5' ⁇ 3' exonuclease is T7 exonuclease.
  • the T7 exonuclease has the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 80% homology therewith.
  • the inventors have found through a large number of experiments that using an exonuclease homologous to T7 exonuclease can reduce the repair efficiency of the NHEJ pathway and improve the repair efficiency of the HDR pathway.
  • the added amount of the site-specific nuclease is 2-50 ng.
  • the inventor obtained the above-mentioned optimal addition amount through a large number of experiments, thus, the repair efficiency of the improved HDR channel can be further filled.
  • the site-specific nuclease is selected from endonucleases, specifically, the site-specific nuclease can be selected from clustered regularly interspaced short palindromic repeats, transcription activation-like effectors At least one of nuclease, zinc finger nuclease, homing endonuclease, and restriction endonuclease.
  • the above-mentioned site-specific nucleases can be used to produce DSBs according to requirements, and the specific types are not limited.
  • the specific nucleases for the above sites can be selected from endonucleases such as clustered regularly interspaced short palindromic repeats, transcription activator-like effector nucleases, zinc finger nucleases, and homing endonucleases. At least one of them may also be a nuclease modified on the basis of any of the above-mentioned site-specific nucleases (for example, introducing a point mutation), and the specific type is not limited.
  • the gRNA includes at least one selected from crRNA/tracrRNA, sgRNA, and pegRNA.
  • the guide RNA in the gene editing system, as long as the guide RNA is capable of forming a complex with the site-specific nuclease and can target the complex to the target due to certain complementarity with the target sequence
  • the sequence is enough, and at least one of the above-mentioned guide RNAs (the guide RNAs in the present invention include traditional guide RNAs and guide RNAs optimized and improved on traditional guide RNA sequences) can be used, wherein the specific type is not limited.
  • the inventors found through experiments that by introducing end protection means to protect the end of the donor DNA as a replication template for the HDR pathway, the donor DNA can be stably present in the cell before editing begins, further improving the efficiency of gene editing, and making the editing efficiency Significantly increased.
  • the above-mentioned donor DNA used in the present invention has higher HDR pathway repair efficiency and HDR/NHEJ ratio.
  • the technical route of the gene editing system includes homologous recombination mediated by an oligonucleotide editing template, single base editing, guided editing, and homologous recombination mediated by a double-stranded long-chain nucleic acid editing template One of.
  • the DSB is produced by the CRISPR/Cas9 system, and the exonuclease is enriched by the MS2 recruitment system, wherein the exonuclease is T7 bacteriophage exonuclease, T5 phage exonuclease (the amino acid sequence is shown in SEQ ID NO: 2 ), lambda bacteriophage exonuclease (as shown in SEQ ID NO: 3), Escherichia coli exonuclease DCLRE1B (as shown in SEQ ID NO: 4), Escherichia coli exonuclease RecE (amino acid The sequence is shown in SEQ ID NO: 5) or human exonuclease EXO1 (amino acid sequence is shown in SEQ ID NO: 6), and the amino acid sequence of T7 bacteriophage exonuclease is shown in SEQ ID NO: 1.
  • the Cas9/sgRNA expression vector and the MCP-exonuclease fusion protein expression vector can be constructed using conventional techniques in the art, for example, see “Gibson, D.G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6,343 -345, doi:10.1038/nmeth.1318(2009)” and "Potapov, V. et al. Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4DNA Ligase and Application to DNA Assembly. ACS Synth Biol 7, 2665-2674, do i :10.1021/acssynbio.8b00333 (2016)”.
  • the repair efficiency of the HDR pathway is shown in Figure 2, where the abscissa indicates the species source of the exonuclease, and all the exonucleases form fusion proteins with MS2, from left to right: no exonuclease negative control, T7 phage Exonuclease, T5 bacteriophage exonuclease, lambda bacteriophage exonuclease, E. coli exonuclease DCLRE1B, E. coli exonuclease RecE and human exonuclease EXO1 active domain; ordinate indicates repair efficiency.
  • the exonuclease is T7 exonuclease.
  • different donor DNAs are selected for experiments, that is, unmodified linear double-stranded DNA, sulfo Phosphodiester bond (PS)-modified linear double-stranded DNA (nPS refers to the 5' end of linear double-stranded DNA modified by n consecutive phosphorothioate bonds) and circular double-stranded DNA in the form of HMEJ were used as donors.
  • PS sulfo Phosphodiester bond
  • nPS refers to the 5' end of linear double-stranded DNA modified by n consecutive phosphorothioate bonds
  • circular double-stranded DNA in the form of HMEJ were used as donors.
  • the HMEJ double-stranded DNA donor is used to introduce consecutive three-base mutations at different positions within 100 bp from the cutting site (nBP refers to the distance between the introduced mutation position and the Cas9 cutting site by n base pairs).
  • nBP refers to the distance between the introduced mutation position and the Cas9 cutting site by n base pairs.
  • the gene editing efficiency was characterized by next-generation sequencing analysis, and the rest of the steps were the same as in Example 1.
  • Donor DNA was not used in this example, only Cas9/sgRNA and T7 exonuclease were introduced as the experimental group (i.e., T7+), and samples were taken at different time points after transfection, and single samples were analyzed by enzyme digestion-fluorescent quantitative PCR method. stranded DNA is detected, and the control uses MCP protein instead of T7 exonuclease (being T7-) (in this embodiment, a 6-well cell culture plate is used, and during transfection, the contents of Cas9/sgRNA and T7 exonuclease are respectively 500ng, 1500ng, PEI dosage is 12 ⁇ L).
  • the results showed that within a period of time after transfection, the total amount of single-stranded DNA near the DSB of the sample added with T7 exonuclease was significantly higher than that of the control group.
  • HMEJ double-stranded DNA donors were used to edit different sites in the cell genome, and the gene editing efficiency was analyzed and characterized by next-generation sequencing (refer to Example 3).
  • the content of Cas9/sgRNA was 20ng, and other steps were the same as in Example 1.
  • the analysis results are shown in Figure 25-30.
  • This embodiment is an extended application of Embodiment 5.
  • the donor DNA with the split-sfGFP tag introduced at the C-terminal of the NPM1 protein that is, NPM1 WT
  • the donor DNA with the pathogenic +4 mutation and the FlAsH short peptide tag introduced at the C-terminal of the NPM1 protein that is, the NPM1 mut
  • the Cas9/sgRNA content is 50ng
  • the T7 exonuclease content is 80ng
  • the donor DNA is 100ng.
  • Other steps are the same as in Example 5.
  • Example 16 Study on the effect of polyQ structure length of ATXN2 protein on protein function by means of gene editing

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Abstract

Utilisation d'une exonucléase dans le sens 5' vers 3' dans un système d'édition génique, et système d'édition génique, et procédé d'édition génique. L'exonucléase dans le sens 5' vers 3' et une nucléase spécifique de site dans le système d'édition génique sont dans un état de non-fusion ; et le système d'édition génique comprend la nucléase spécifique de site, l'exonucléase dans le sens 5' vers 3', et un ADN donneur, l'exonucléase dans le sens 5' vers 3' et la nucléase spécifique de site étant dans un état de non-fusion.
PCT/CN2023/079632 2022-03-03 2023-03-03 Utilisation d'une exonucléase dans le sens 5' vers 3' dans un système d'édition génique, et système d'édition génique, et procédé d'édition génique WO2023165613A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020236645A1 (fr) * 2019-05-17 2020-11-26 Beth Israel Deaconess Medical Center, Inc. Compositions et méthodes pour la réparation dirigée d'homologie
US20200407754A1 (en) * 2019-06-25 2020-12-31 Inari Agriculture, Inc. Homology dependent repair genome editing
CN113025597A (zh) * 2019-12-24 2021-06-25 中国科学院微生物研究所 改进的基因组编辑系统
CN113481184A (zh) * 2021-08-06 2021-10-08 北京大学 融合蛋白以及其使用方法
US20210403922A1 (en) * 2019-01-07 2021-12-30 Crisp-Hr Therapeutics, Inc. Non-toxic cas9 enzyme and application thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210403922A1 (en) * 2019-01-07 2021-12-30 Crisp-Hr Therapeutics, Inc. Non-toxic cas9 enzyme and application thereof
WO2020236645A1 (fr) * 2019-05-17 2020-11-26 Beth Israel Deaconess Medical Center, Inc. Compositions et méthodes pour la réparation dirigée d'homologie
US20200407754A1 (en) * 2019-06-25 2020-12-31 Inari Agriculture, Inc. Homology dependent repair genome editing
CN113025597A (zh) * 2019-12-24 2021-06-25 中国科学院微生物研究所 改进的基因组编辑系统
CN113481184A (zh) * 2021-08-06 2021-10-08 北京大学 融合蛋白以及其使用方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
QIANWEI ZHANG; KANGQUAN YIN; GUANWEN LIU; SHENGNAN LI; MENGOU LI; JIN-LONG QIU: "Fusing T5 Exonuclease with Cas9 and Cas12a Increases the Frequency and Size of Deletion at Target Sites", SCIENCE CHINA LIFE SCIENCES, ZHONGGUO KEXUE ZAZHISHE, CHINA, vol. 63, no. 12, 6 May 2020 (2020-05-06), China , pages 1918 - 1927, XP009548348, ISSN: 1674-7305 *

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