WO2021224506A1 - Agent d'amélioration de la réparation dirigée par l'homologie de système crispr-cas - Google Patents

Agent d'amélioration de la réparation dirigée par l'homologie de système crispr-cas Download PDF

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Publication number
WO2021224506A1
WO2021224506A1 PCT/EP2021/062363 EP2021062363W WO2021224506A1 WO 2021224506 A1 WO2021224506 A1 WO 2021224506A1 EP 2021062363 W EP2021062363 W EP 2021062363W WO 2021224506 A1 WO2021224506 A1 WO 2021224506A1
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crispr
cas system
previous
protein
hdr
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PCT/EP2021/062363
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English (en)
Inventor
Marc GÜELL CARGOL
Avencia SÁNCHEZ-MEJÍAS GARCÍA
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Universitat Pompeu Fabra
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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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • 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
    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/01—Methyltransferases (2.1.1)
    • C12Y201/01043—Histone-lysine N-methyltransferase (2.1.1.43)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • 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 present invention refers to the genetic engineering field.
  • the present invention refers to a CRISPR-Cas system, suitable for gene edition, which comprises: a) at least a guide sequence capable of hybridizing to a target sequence in a eukaryotic cells, b) at least an effector protein, or a nucleic acid encoding the effector protein, with nuclease or nickase activity, and c) catalytic domain of a chromatin remodelling protein coupled to the effector protein.
  • Genome editing is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike early genetic engineering techniques that randomly inserts genetic material into a host genome, genome editing targets the insertions to site specific locations.
  • Genome editing can be achieved by using CRISPR-Cas system, wherein a small piece of RNA with a short "guide” sequence attaches (binds) to a specific target sequence of DNA in a genome.
  • the RNA also binds to the Cas enzyme.
  • the modified RNA is used to recognize the DNA sequence, and the Cas enzyme cuts the DNA at the targeted location.
  • Cas9 is the enzyme that is used most often, other enzymes (for example Cpfl) can also be used.
  • homology directed repair is a mechanism used by the cells to repair DNA cuts.
  • the most common form of HDR is homologous recombination.
  • the HDR mechanism can only be used by the cell when there is a homologous piece of DNA present in the nucleus, mostly in G2 and S phase of the cell cycle.
  • the cell machinery resolves the event by next homologous end joining, which has unpredictable and deleterious effects. To do precise gene editing this cut needs to be repaired by homologous recombination which is very inefficient.
  • the objective of the present invention is to increase the efficiency of HDR mechanism in the context of gene editing.
  • the present invention solves this problem by providing a strategy for improving the efficiency of the HDR system after CRISPR-Cas nuclease activity on a DNA target. This has been achieved by coupling CRISPR-Cas to specific proteins as explained below.
  • the present invention refers to a CRISPR-Cas system, suitable for gene edition, wherein the efficiency of the HDR system after CRISPR-Cas nuclease activity on a DNA target has been improved.
  • the first embodiment of the present invention refers to a CRISPR-Cas system of the invention comprises: a) at least a guide sequence capable of hybridizing to a target sequence in a eukaryotic cells, b) at least an effector protein, or a nucleic acid encoding the effector protein, with nuclease or nickase activity, and c) catalytic domain of a chromatin remodelling protein coupled to the effector protein.
  • the CRISPR-Cas system further comprises an HDR template.
  • the HDR template may consist of a DNA fragment of variable length with a right and left homology arm and a central region containing the desired edit to introduce in the genome.
  • the HDR template used can be a ssDNA linear or circular, a dsDNA linear or circular or an AAV DNA genome.
  • the effector protein is cas9 or Cpf 1.
  • the chromatin remodelling protein is a member of the PRDM family.
  • the chromatin remodelling protein is a member of the chromatin remodelling protein is selected from the group comprising: PRDM9, PRDMl, PRDM2, PRDM3, PRDM 16 or PRDM6.
  • the chromatin remodelling protein member of the chromatin remodelling protein is PRDM9.
  • the chromatin remodelling protein is coupled in N- or C- configuration.
  • the effector protein and/or the chromatin remodelling protein are wild type proteins. In a preferred embodiment, the effector protein and/or the chromatin remodelling protein are mutant proteins.
  • system further comprises at least one aptamers linked to the guide sequence.
  • system further comprises at least one linker, preferably selected from:
  • system further comprises a MS2 loop linked to the guide sequence.
  • the system further comprises one or more HDR activator and/or one or more NHEJ (Non-homologous end joining) inhibitor fused to the effector protein, or combinations thereof.
  • NHEJ Non-homologous end joining
  • the HDR activator is selected from the group comprising: RAD51, RAD52, DMC1, CtIP, or any combination thereof.
  • the NHEJ inhibitors is selected from the group comprising: E1B55K, E4orf6, 53BP1(DM), Rif, p53, or any combination thereof.
  • the one or more HDR activator and/or one or more NHEJ inhibitor proteins is fused to the effector protein.
  • the second embodiment of the present invention refers to a method for gene edition which comprises the use of the CRISPR-Cas system defined above.
  • FIG. 1 Cas9 PDMR9 fusion.
  • FIG. 1 Traffic light reporter system for HDR and NHEJ.
  • FIG. 1 HDR enhancement in Cas9 fusion proteins by additional factors.
  • PRDM9 catalytic domain together with a flexible linker was purchased from Twist Bioscience.
  • PRDM9 was cloned into a Cas9-expression vector Addgene #41815 using isothermal assembly following standard protocols.
  • Dead (D10A and H840A) and nickase (D10 A) Cas9 mutations as well as PRDM9 catalytic mutation (G172A) were introduced by site directed mutagenesis (New England Biolabs).
  • the collection of HDR enhancing factors fused to MS2 protein were constructed based on the pcDNATM3.1 vector backbone (Thermo Fisher).
  • the following expression vectors were amplified by PCR: RAD51 (Addgene #41815), CtIP (Addgene #109403), Ad4orf2B (Addgene #64221), Ad4E4orf6 (Addgene #64221), 53BP1-DN (Addgene #131045), D-sup (Addgene #90019) and DMC1 (CRG CDS collection) were amplified by PCR. Similarly, MS2 expressing vector (Addgene #61423) was also amplified by PCR. HDR enhancing factors and MS2 tag were cloning between Esp3I sites by Golden Gate Assembly.
  • MS2-UL12 a gBlock of MS2-UL12 was ordered to Twist Bioscience and cloned into pcDNATM3.1 vector backbone by Golden Gate Assembly.
  • MS2-RAD52 was ordered as a plasmid vector to Twist Bioscience.
  • gRNAs were cloned into sg-RNA(MS2) cloning vector (Addgene #61424) between Bbsl sites using standard cloning methods.
  • MS2-RAD52 was ordered as a plasmid vector to Twist Bioscience.
  • gRNAs were cloned into sg-RNA(MS2) cloning vector (Addgene #61424) between Bbsl sites using standard cloning methods.
  • MS2 sg-RNA(MS2) cloning vector
  • Hek293T cells were infected with TLR lentivirus at a 0,2 multiplicity of infection (MOI) to generate the TLR cell line. After infection cells were selected with puromycin following standard protocols for 2 weeks before being used for subsequent assays. Hek293T cell line containing pT4 SMN1 2/2 emGFP was generated by PEI mediated transfection of SB100X and pT4 SMN1 2/2 emGFP DNA constructs, followed by single clone expansion and PCR genotyping. A positive clone was selected and expanded and used for subsequent assays.
  • MOI multiplicity of infection
  • TLR cell line and C2C12 cell line were cultured at 37°C in a 5% CO 2 incubator with Dulbecco’s modified eagle medium (DMEM), supplemented with high glucose (Gibco, Therm Fisher), 10% Fetal Bovine Serum (FBS), 2 mM glutamine and 100 U penicillin/0.1 mg/mL streptomycin.
  • DMEM Dulbecco modified eagle medium
  • FBS Fetal Bovine Serum
  • streptomycin 100 U penicillin/0.1 mg/mL streptomycin.
  • FiPSC-Ctrll-Ep6F-5 cell line was purchased from the Biobank of the Barcelona Centre for Regenerative Medicine (CMRB). Cell’s transfection experiments were performed with Polyethyleneimine (PEI, Thermo Fisher Scientific) at 1:3 DNA-PEI ratio in OptiMem. Cells were seeded the day before to achieve 70% confluence on transfection day (usually 120.000 cells in adherent p24 well plate). Plasmid molar ratio was 1 of Cas9 or Cas9-PRDM9: 3 gRNA: 3 HDR template using 0,089 pmols of Cas9 or Cas9-PRDM9 for a p24 well plate.
  • Cas9 or Cas9-PRDM9 3 gRNA: 3 HDR template using 0,089 pmols of Cas9 or Cas9-PRDM9 for a p24 well plate.
  • Electroporation of C2C12 and iPS cells was performed according to the manufacturer’s instructions with Lonza 4D-Nucleofector System with CD-137 program (C2C12). In the case of the iPS the parameters P3 Primary Cell 4D-Nucleofector were followed and the program CM-113 was applied.
  • DNA contains wild-type and edited DNA molecules, which were amplified together using the same pairs of primers.
  • the two PCR reactions were performed with KAPA HiFi DNA Polymerase following manufacturer protocol: lOOng DNA, 0.3 mM of forward and reverse primers in a final reaction volume of 25 pi.
  • Primers included sequencing adapters to their 3’ -ends, adding them to both termini of PCR products that amplified genomic DNA.
  • the primers used appended dual sample indexes and flow cell adapters.
  • PCR products were purified with a PCR purification kit (Qiagen) and quantified using the QuBIT dsDNA High Sensitivity Assay kit and the QuBIT 2.0 fluorometer according to the manufacturer’s instructions (Life Technologies) before preparing the sequencing reaction.
  • the inventors of the present invention first constructed a C-terminal fusion of S. pyogenes cas9 and PDRM9 and tested recombination efficiency with one split GFP reporter, both as episomal DNA and in cells containing the split GFP reporter in the genome (Figure 1A).
  • This library ( Figure 4A) consist of multiple selected rad51 orthologues and rad52 which are key in HDR, in addition to other HDR associated proteins such as CtIP and MREll, important in the resection of DNA after a DSB that have HDR enhancer activity when fussed to Cas9, as well as other proteins such as adenovirus 4 proteins E1B55K and E4orf6 which inhibit the competing pathway NHEJ.
  • Expression of i53 also inhibit NHEJ, by suppressing 53BP1, a key regulator of DSB repair pathway choice in eukaryotic cells and functions to favor NHEJ over HDR. Additionally, we tested the protein Dsup from radiation resistant organism Tardigrade.
  • DMC1 a meiosis specific protein implicated in the repair of DSB using the homologous chromosome as the template for repairing the break.
  • the pooled library and chimeric Cas9 protein will be cloned into a lentiviral vector and transduced into a population of cells containing the HDR reporter. After transfection with gRNA and template, FACS sorting will be used to enrich for cells that underwent HDR. This population will be analyzed to find overrepresented library members which could be potential activators of HDR. Genotyping will be performed using a specific DNA barcode that will have been added previously to each library member.

Abstract

La présente invention concerne un système CRISPR-Cas, approprié pour l'édition génique, qui comprend: a) au moins une séquence guide capable de s'hybrider à une séquence cible dans des cellules eucaryotes, b) au moins une protéine effectrice, ou un acide nucléique codant pour la protéine effectrice, avec une activité nucléase ou nickase, et c) le domaine catalytique d'une protéine de remodelage de la chromatine couplée à la protéine effectrice.
PCT/EP2021/062363 2020-05-08 2021-05-10 Agent d'amélioration de la réparation dirigée par l'homologie de système crispr-cas WO2021224506A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114438036A (zh) * 2021-12-03 2022-05-06 中国人民解放军军事科学院军事医学研究院 一种促进干细胞向红系细胞定向分化与成熟的方法与应用

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US9790490B2 (en) * 2015-06-18 2017-10-17 The Broad Institute Inc. CRISPR enzymes and systems
WO2017184786A1 (fr) * 2016-04-19 2017-10-26 The Broad Institute Inc. Complexes cpf1 à activité d'indel réduite
WO2018005873A1 (fr) * 2016-06-29 2018-01-04 The Broad Institute Inc. Systèmes crispr-cas ayant un domaine de déstabilisation
WO2018035387A1 (fr) * 2016-08-17 2018-02-22 The Broad Institute, Inc. Nouveaux systèmes et enzymes crispr
US20180298392A1 (en) * 2014-11-07 2018-10-18 Editas Medicine, Inc. Methods for improving crispr/cas-mediated genome-editing
EP3428274A1 (fr) * 2017-07-11 2019-01-16 Sigma Aldrich Co. LLC Utilisation de domaines de protéines interagissant avec des nucléosomes. pour améliorer la modification ciblée du génome
WO2019126762A2 (fr) * 2017-12-22 2019-06-27 The Broad Institute, Inc. Systèmes cas12a, procédés et compositions d'édition ciblée de bases d'arn
US20190233805A1 (en) * 2017-10-04 2019-08-01 The Regents Of The University Of California Targetable proteins for epigenetic modification and methods for use thereof
US20190345490A1 (en) * 2016-03-25 2019-11-14 Editas Medicine, Inc. Genome editing systems comprising repair-modulating enzyme molecules and methods of their use
US20200080112A1 (en) * 2016-08-17 2020-03-12 The Broad Institute, Inc. Novel crispr enzymes and systems

Patent Citations (10)

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US20180298392A1 (en) * 2014-11-07 2018-10-18 Editas Medicine, Inc. Methods for improving crispr/cas-mediated genome-editing
US9790490B2 (en) * 2015-06-18 2017-10-17 The Broad Institute Inc. CRISPR enzymes and systems
US20190345490A1 (en) * 2016-03-25 2019-11-14 Editas Medicine, Inc. Genome editing systems comprising repair-modulating enzyme molecules and methods of their use
WO2017184786A1 (fr) * 2016-04-19 2017-10-26 The Broad Institute Inc. Complexes cpf1 à activité d'indel réduite
WO2018005873A1 (fr) * 2016-06-29 2018-01-04 The Broad Institute Inc. Systèmes crispr-cas ayant un domaine de déstabilisation
WO2018035387A1 (fr) * 2016-08-17 2018-02-22 The Broad Institute, Inc. Nouveaux systèmes et enzymes crispr
US20200080112A1 (en) * 2016-08-17 2020-03-12 The Broad Institute, Inc. Novel crispr enzymes and systems
EP3428274A1 (fr) * 2017-07-11 2019-01-16 Sigma Aldrich Co. LLC Utilisation de domaines de protéines interagissant avec des nucléosomes. pour améliorer la modification ciblée du génome
US20190233805A1 (en) * 2017-10-04 2019-08-01 The Regents Of The University Of California Targetable proteins for epigenetic modification and methods for use thereof
WO2019126762A2 (fr) * 2017-12-22 2019-06-27 The Broad Institute, Inc. Systèmes cas12a, procédés et compositions d'édition ciblée de bases d'arn

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114438036A (zh) * 2021-12-03 2022-05-06 中国人民解放军军事科学院军事医学研究院 一种促进干细胞向红系细胞定向分化与成熟的方法与应用
CN114438036B (zh) * 2021-12-03 2023-08-18 中国人民解放军军事科学院军事医学研究院 一种促进干细胞向红系细胞定向分化与成熟的方法与应用

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