WO2021121321A1 - 一种提高基因编辑效率的融合蛋白及其应用 - Google Patents

一种提高基因编辑效率的融合蛋白及其应用 Download PDF

Info

Publication number
WO2021121321A1
WO2021121321A1 PCT/CN2020/137239 CN2020137239W WO2021121321A1 WO 2021121321 A1 WO2021121321 A1 WO 2021121321A1 CN 2020137239 W CN2020137239 W CN 2020137239W WO 2021121321 A1 WO2021121321 A1 WO 2021121321A1
Authority
WO
WIPO (PCT)
Prior art keywords
fusion protein
cells
sequence
seq
be4max
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2020/137239
Other languages
English (en)
French (fr)
Chinese (zh)
Inventor
李大力
张晓辉
刘明耀
朱碧云
陈亮
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
East China Normal University
Bioray Laboratories Inc
Original Assignee
East China Normal University
Bioray Laboratories Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN201911310969.8A external-priority patent/CN112979821B/zh
Priority claimed from CN201911312544.0A external-priority patent/CN112979823B/zh
Priority claimed from CN201911312537.0A external-priority patent/CN112979822B/zh
Application filed by East China Normal University, Bioray Laboratories Inc filed Critical East China Normal University
Priority to EP20903960.1A priority Critical patent/EP4079765A4/en
Priority to JP2022538379A priority patent/JP7606176B2/ja
Publication of WO2021121321A1 publication Critical patent/WO2021121321A1/zh
Priority to US17/843,462 priority patent/US20220364072A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • 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
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • 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
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04001—Cytosine deaminase (3.5.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04004—Adenosine deaminase (3.5.4.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04005—Cytidine deaminase (3.5.4.5)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • 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
    • 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
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • 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]
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites

Definitions

  • the invention relates to the field of biotechnology, in particular to a fusion protein for improving gene editing efficiency and its application.
  • CRISPR/Cas9 derived from Streptococcus pyogenes spCas9 to use NGG as the PAM (spacer sequence precursor adjacent motif) and recognizes and specifically binds to the single base from C to T or G to A upstream of NGG. Base mutation.
  • BE3 base editor 3
  • UGI uracil glycosidase inhibitor
  • ⁇ -hemoglobinopathy such as ⁇ -thalassemia and sickle cell disease (SCD) is caused by mutations in the HBB gene encoding ⁇ -hemoglobin.
  • SCD sickle cell disease
  • HPFH hereditary fetal hemoglobinopathy
  • HPFH hereditary fetal hemoglobinopathy
  • the high expression of gamma-globin in adult patients can alleviate the disease phenotype caused by beta hemoglobin mutations. It has been reported that deleting 13bp of the promoter region of HBG1/2 with CRISPR/Cas9 can activate the expression of ⁇ -globulin, thereby alleviating or treating thalassemia disease, which is an effective treatment strategy.
  • a heterozygous point mutation at position -117 in the HBG1/2 promoter region produced 10-20% of fetal hemoglobin (HbF) expression.
  • the mechanism is -117G>A
  • the mutation destroys the binding site of the transcription repressor BCL11A.
  • the purpose of the present invention is to provide a fusion protein that improves gene editing efficiency and its application.
  • the present invention provides a fusion protein that improves gene editing efficiency, including a single-stranded DNA binding protein functional domain, nucleoside deaminase and nuclease.
  • connection sequence of the fusion protein is: the nucleoside deaminase is located at the N-terminus or the C-terminus of the nuclease, and the single-stranded DNA binding protein functional domain is located at the nucleoside deaminase and the nucleoside deaminase.
  • the nucleoside deaminase is located at the N-terminus of the nuclease
  • the single-stranded DNA binding protein functional domain is located between the nucleoside deaminase and the nuclease.
  • the single-stranded DNA binding protein includes a sequence-specific single-stranded DNA binding protein, and/or a non-sequence-specific single-stranded DNA binding protein, preferably, a non-sequence-specific single-stranded DNA binding protein,
  • the non-sequence-specific single-stranded DNA binding protein is selected from RPA70 (subunit 70 of human replication protein A), RPA32 (subunit 32 of human replication protein A), BRCA2 (breast cancer No. 2 gene), hnRNPK (Heterogeneous nuclear ribonucleoprotein K), PUF60 (poly-U binding splicing factor 60KDa) and Rad51 (a homologous recombination repair protein) any one or more;
  • sequence-specific single-stranded DNA binding protein is selected from any one of TEBP (telomere binding protein), Teb1 (a constituent protein of telomerase) and POT1 (human telomere protective protein 1) or Any number
  • the single-stranded DNA-binding protein functional domain includes at least one of the following four domains (any one, any two, any three or all four) or the following four domains have a single Partial polypeptide fragments with chain DNA binding function and any combination thereof: OB folding (oligonucleotide/oligosaccharide/oligopeptide binding folding), KH domain (K homology domain), RRMS of the single-stranded DNA binding protein (RNA recognition motif), whirly domains;
  • the single-stranded DNA binding protein functional domain includes the DNA binding domain (DBD) of Rad51. More preferably, the amino acid sequence of the DNA binding domain of Rad51 includes the sequence shown in SEQ ID No. 1, and more Preferably, the coding sequence of the DNA binding domain of Rad51 includes the sequence shown in SEQ ID No. 2;
  • the amino acid sequence of the DNA binding domain of RPA70 includes the sequence shown in SEQ ID No. 11, and more preferably, the coding sequence of the DNA binding domain of RPA70 includes the sequence shown in SEQ ID No. 12.
  • the deaminase includes cytosine deaminase (APOBEC) and/or adenosine deaminase, preferably, cytosine deaminase, and the cytosine deaminase can be derived from different organisms. body,
  • the cytosine deaminase includes rat-derived cytosine deaminase.
  • the rat-derived cytosine deaminase amino acid sequence includes SEQ ID No. 3
  • the sequence, more preferably, the coding sequence of the rat-derived cytosine deaminase includes the sequence shown in SEQ ID No. 4;
  • the cytosine deaminase includes human-derived cytosine deaminase APOBEC3A.
  • the amino acid sequence of the human-derived cytosine deaminase APOBEC3A includes SEQ ID No. 13 More preferably, the coding sequence of the cytosine deaminase APOBEC3A includes the sequence shown in SEQ ID No. 14;
  • the cytosine deaminase includes a mutant of cytosine deaminase APOBEC3A, and the cytosine deaminase APOBEC3A mutant is the cytosine deaminase APOBEC3A at position 57 (from the start Codon) asparagine (N or Asn) is mutated to glycine (G or Gly), preferably, the cytosine deaminase APOBEC3A is derived from humans, more preferably, the amino acid sequence of the cytosine deaminase APOBEC3A includes The sequence shown in SEQ ID No.
  • the coding sequence of the cytosine deaminase APOBEC3A includes the sequence shown in SEQ ID No. 14; the amino acid sequence of the cytosine deaminase APOBEC3A mutant includes SEQ ID No.
  • the sequence shown in .15, more preferably, the coding sequence of the cytosine deaminase APOBEC3A includes the sequence shown in SEQ ID No. 16;
  • the nuclease is selected from one or more of Cas9, Cas3, Cas8a, Cas8b, Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1, Cpf1; preferably
  • the nuclease is Cas9; more preferably, the Cas9 is selected from Cas9 derived from Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus pyogenes or Streptococcus thermophilus, more preferably, the Cas9 is selected from Cas9 Mutants VQR-spCas9, VRER-spCas9, spCas9n, more preferably, spCas9n, more preferably, the amino acid sequence of spCas9n includes the sequence shown in SEQ ID No. 5, and more preferably, the coding sequence of spCas9n includes
  • NLS Nuclear Localization Signal
  • the NLS is located at at least one end (C-terminal and/or N-terminal) of the fusion protein; more preferably, the amino acid sequence of the NLS includes The sequence shown in SEQ ID No. 7, and more preferably, the coding sequence of the NLS includes the sequence shown in SEQ ID No. 8;
  • the fusion protein also includes UGI (uracil glycosidase inhibitor).
  • UGI uracil glycosidase inhibitor
  • the UGI is located at at least one end (C-terminal and/or N-terminal) of the fusion protein; more preferably, the amino acid sequence of the UGI It includes the sequence shown in SEQ ID No. 9, and more preferably, the coding sequence of the UGI includes the sequence shown in SEQ ID No. 10, and more preferably, the UGI is more than two copies.
  • the present invention also provides any one of the following A)-C) biological materials:
  • a gene encoding any one of the above-mentioned fusion proteins is DNA or RNA (such as mRNA);
  • Immune cells such as T cells
  • hematopoietic stem cells such as T cells
  • bone marrow cells such as red blood cells, preferably red blood cell precursor cells or hematopoietic stem cells.
  • the present invention also provides a sgRNA for gene-editing a target gene in a cell, and the target sequence of the sgRNA includes at least one of SEQ ID No. 17-36,
  • the cells are T cells, hematopoietic stem cells, bone marrow cells or red blood cells, more preferably, red blood cell precursor cells or hematopoietic stem cells,
  • the target genes are HBG1 and HBG2 promoter regions (specifically, the G at position -117 is edited as A, and the G at position -117 is the 14th G from the left in the promoter region CCAGCCTTGCCTTGACCAATAGCC).
  • the present invention also provides a single-base gene editing system, comprising any one of the above fusion protein or the biological material and sgRNA, and the sgRNA guides the purpose of the fusion protein to the target cell.
  • Single-base gene editing of genes comprising any one of the above fusion protein or the biological material and sgRNA, and the sgRNA guides the purpose of the fusion protein to the target cell.
  • the target sequence of the sgRNA includes at least one of SEQ ID No. 17-36;
  • the cells are T cells, hematopoietic stem cells, bone marrow cells, red blood cells, or red blood cell precursor cells,
  • the target genes are HBG1 and HBG2 promoter regions.
  • the present invention protects any of the fusion proteins, the biological materials, and the single-base gene editing system in the preparation of gene editing products, disease treatment and/prevention products, animal models, or new plant varieties.
  • the disease is beta hemoglobinopathy
  • the beta hemoglobinopathy includes beta thalassemia and/or sickle cell anemia.
  • the present invention also provides a method for improving the efficiency of single-base gene editing, including the steps of introducing any one of the above fusion proteins and sgRNA into cells and performing gene editing on the target gene.
  • the sgRNA guides the The fusion protein performs single-base gene editing on the target gene.
  • the target sequence of the sgRNA includes at least one of SEQ ID No. 17-36;
  • the cells are T cells, hematopoietic stem cells, bone marrow cells, red blood cells, or red blood cell precursor cells;
  • the target genes are HBG1 and HBG2 promoter regions.
  • the present invention also provides a method for constructing an animal model of a disease, including the steps of introducing any one of the above-mentioned fusion proteins and sgRNA into animal cells and performing gene editing on the target gene;
  • the target sequence of the sgRNA includes at least one of SEQ ID No. 17-36; more preferably, the target sequence of the sgRNA includes the sequence shown in SEQ ID No. 36, and the target gene includes the DMD gene ;
  • the animal is a mammal, more preferably, the mammal is a rat or a mouse, more preferably a mouse;
  • the cell is an embryonic cell
  • the method of introduction is one of vector transformation, microinjection, transfection, lipofection, heat shock, electroporation, transduction, gene gun, DEAE-dextran-mediated transfer, or Any combination, more preferably, microinjection;
  • the introduction is carried out using the mRNA of any one of the above-mentioned fusion proteins and the sgRNA,
  • the concentration of the mRNA of any one of the above fusion proteins used in the introduction is 1-1000ng/ ⁇ L, more preferably, 10-600ng/ ⁇ L, more preferably, 50-150ng/ ⁇ L, more preferably, 100ng / ⁇ L
  • the concentration of the introduced sgRNA used is 1-1000ng/ ⁇ L, more preferably, 10-600ng/ ⁇ L, more preferably, 150-250ng/ ⁇ L, more preferably, 200ng/ ⁇ L
  • the concentration ratio of the mRNA of any of the above fusion proteins used in the introduction to the sgRNA used in the introduction is 1:(5-1), more preferably, 1:(4-1.5), More preferably, 1:(3-1.8), more preferably, 1:2.
  • the invention protects the application of the animal model obtained by the method in drug screening, disease treatment effect evaluation or disease treatment mechanism research.
  • the present invention provides a product for the treatment and/or prevention of ⁇ -hemoglobinopathy, comprising: a delivery vector of the gene and sgRNA described in A) above,
  • the sgRNA guides the fusion protein to affect the HBG1 and HBG2 promoter regions in the target cell (specifically, the G at position -117 is edited as A, and the G at position -117 is the 14th position from the left in the promoter region CCAGCCTTGCCTTGACCAATAGCC G) Perform single-base gene editing;
  • the target sequence of the sgRNA includes the sequence shown in SEQ ID No. 35;
  • the beta hemoglobinopathy includes beta thalassemia and/or sickle cell anemia
  • the cells are T cells, hematopoietic stem cells, bone marrow cells or red blood cells, more preferably, red blood cell precursor cells or hematopoietic stem cells,
  • the target genes are HBG1 and HBG2 promoter regions (specifically, the G at position -117 is edited as A, and the G at position -117 is the 14th G from the left in the promoter region CCAGCCTTGCCTTGACCAATAGCC).
  • the delivery vector includes a viral vector and/or a non-viral vector;
  • the viral vector includes an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, a retroviral vector, and/or an oncolytic viral vector ,
  • the non-viral vector includes cationic polymer, plasmid vector and/or liposome;
  • the delivery vehicle includes a lentiviral vector.
  • the homology with the sequence described in this application is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or More than 99% of the sequence, and/or the sequence after substitution, deletion or insertion of amino acid residues or nucleotides based on the sequence described in this application, and a sequence with the same or similar functions as the sequence used in this application , Are all within the scope of protection of this application.
  • the present invention uses CBEs (pyrimidine base conversion technology) in the process of CG to TA base conversion.
  • Nucleoside deaminase such as cytosine deaminase, uses single-stranded DNA as a substrate for deamination.
  • the single-stranded DNA binding protein functional domain is fused to the nuclease fusion protein, which greatly increases the chance of single-stranded DNA being exposed to nucleoside deaminase, thereby significantly improving the base editing efficiency.
  • the present invention screened the functional domains of 10 non-sequence-preferred single-stranded DNA binding proteins to be fused with BE4max, and found that a single-stranded DNA binding functional domain (1-114AA) derived from human Rad51 was fused in the middle of Apobec1 and Cas9n. The highest efficiency improvement was produced, named hyBE4max. Compared with BE4max, the C-G to T-A editing efficiency of hyBE4max is increased by 16 times, especially for sites close to the PAM region, while maintaining low indels (insertions or deletions).
  • the present invention has made a breakthrough improvement on single-base gene editing technology, and can greatly promote its application in gene editing, gene therapy, cell therapy, animal model making, crop genetic breeding and the like.
  • the present invention takes ⁇ -hemoglobinopathy as an example.
  • A3A-BE4max, hyeA3A-BE4max targets HBG1 and HBG2 (hereinafter referred to as HBG1/2) promoter regions closer to PAM region -117 It can more accurately and efficiently target -117 to generate G to A mutations, thereby activating the expression of ⁇ -globin, providing a more precise and efficient treatment strategy for clinical treatment of ⁇ -hemoglobinopathy.
  • HBG1/2 HBG1 and HBG2
  • the present invention applies hyA3A-BE4max to the production of mouse disease animal models. Compared with A3A-BE4max, hyA3A-BE4max targets C to T mutations near the PAM region to produce disease animal models. Therefore, the present invention provides a A new type of platform for efficiently producing disease animal models will greatly promote the production process of different animal models.
  • FIG. 1 is a schematic diagram of the fusion of different single-stranded DNA binding protein functional domains with BE4max.
  • NLS is a nuclear localization signal (its amino acid sequence is shown in SEQ ID No. 7, and its coding sequence is shown in SEQ ID No. 8), and rA1 is cytidine deaminase APOBEC1 (its amino acid sequence is shown in SEQ ID No. 3).
  • the coding sequence is shown in SEQ ID No. 4
  • spCas9n is Cas9n derived from Streptococcus pyogenes (its amino acid sequence is shown in SEQ ID No. 5, and the coding sequence is shown in SEQ ID No.
  • UGI is a uracil glycosidase inhibitor (its amino acid sequence is shown in SEQ ID No. 9 and the coding sequence is shown in SEQ ID No. 10), and SSDBD is a single-stranded DNA binding protein functional domain.
  • Figure 2 shows the comparison of C to T base editing efficiency (ie, the ordinate, in %) achieved by hyBE4max and BE4max on 8 targets on 293T.
  • Figure 3 is a comparison of the average C to T base editing efficiency (ie, the ordinate, in %) produced by hyBE4max and BE4max on 8 targets on 293T.
  • Figure 4 is a comparison of the base editing efficiency (ie, the ordinate, the unit is %) of indels generated by hyBE4max and BE4max on 8 targets on 293T.
  • FIG. 5 is a schematic diagram of the structure of the fusion proteins A3A-BE4max and hyA3A-BE4max.
  • hA3A is a human-derived cytidine deaminase APOBEC3A (its amino acid sequence is shown in SEQ ID No. 13, and the coding sequence is shown in SEQ ID No. 14), and NLS, spCas9n and UGI are the same as in Fig. 1.
  • Figure 6 is a comparison of the C to T base editing efficiency (ie, the ordinate, in %) achieved by hyA3A-BE4max and A3A-BE4max on 8 endogenous targets on 293T.
  • Fig. 7 is a comparison of the average C to T base editing efficiency (i.e. the ordinate, the unit is %) achieved by hyA3A-BE4max and A3A-BE4max on 8 endogenous targets on 293T.
  • Fig. 8 is a comparison of the base editing efficiency (ie, the ordinate, the unit is %) of indels generated by 8 endogenous targets on 293T between hyA3A-BE4max and A3A-BE4max.
  • Figure 9 is a schematic diagram of the structure of the fusion proteins eA3A-BE4max and hyeA3A-BE4max.
  • A3A N57G is the N57G mutant of hA3A used in Figure 5 (its amino acid sequence is shown in SEQ ID No. 15, and the coding sequence is shown in SEQ ID No. 16), and NLS, spCas9n and UGI are the same as in Figure 1.
  • Figure 10 is a comparison of the C to T base editing efficiency (i.e. the ordinate, the unit is %) achieved by hyeA3A-BE4max and eA3A-BE4max on 11 endogenous targets on 293T.
  • Figure 11 is a comparison of the average C to T base editing efficiency (i.e. the ordinate, the unit is %) achieved by hyeA3A-BE4max and eA3A-BE4max on 11 endogenous targets on 293T.
  • Figure 12 is a comparison of the base editing efficiency (ie the ordinate, the unit is %) of indels generated by 11 endogenous targets on 293T between hyeA3A-BE4max and eA3A-BE4max.
  • the abscissa C in Figures 2, 3, 5, 6, and 11 represents the position of the C edited as T on the corresponding target sequence.
  • C5 represents the position from the 5'end of the corresponding target sequence.
  • the 5-digit C is edited as the efficiency of T.
  • Figure 13 is a schematic diagram of hyeA3A-BE4max targeting HBG1/2 promoter region -117G.
  • the -117G>A mutation is shown in red
  • the core sequence of the transcription factor BCL11A binding site is represented by a box
  • the PAM sequence is blue.
  • the G>A transformation destroys the transcription repressor BCL11A binding site and activates HUDEP- 2 Expression of HBG1/2 in ( ⁇ G ⁇ ).
  • Figure 14 is a comparison of the C to T base editing efficiency (ie, the ordinate, the unit is %) achieved by targeting the HBG-117G target in HEK293T cells by hyeA3A-BE4max, eA3A-BE4max, A3A-BE4max, and hyA3A-BE4max.
  • Figure 15 is a schematic diagram of the construction of lentiviral vectors Lenti-117G-hyA3A-BE4max-P2A-GFP and Lenti-117G-hyeA3A-BE4max-P2A-GFP.
  • the target sequence of sgRNA is HBG-117G.
  • FIG 16 is a hyeA3A-BE4max targeting and hyA3A-BE4max C HBG-117G targets implemented in HUDEP-2 ( ⁇ G ⁇ ) cells to T bases editing efficiency (i.e. the ordinate, the unit is%) contrast.
  • FIG 17 is a lentivirus infection Lenti-117G-hyeA3A-BE4max- P2A-GFP , and ( ⁇ G ⁇ ) Globin mRNA after cell differentiation Comparative Lenti-117G-hyA3A-BE4max- P2A-GFP in HUDEP-2. Among them, **** means the difference significance level P ⁇ 0.0001.
  • Figure 18 is a schematic diagram of the animal model construction of hyA3A-BE4max targeting Duchenne muscular dystrophy (DMD) gene.
  • DMD Duchenne muscular dystrophy
  • Figure 19 is a comparison of F0 high-throughput sequencing results after microinjection of A3A-BE4max and hyA3A-BE4max.
  • Figure 20 shows the average ratio of Reads containing TAA stop codons in F0 produced by A3A-BE4max and hyA3A-BE4max injections.
  • FIG. 21 shows the F0 mice produced by immunofluorescence staining to detect the expression of dystrophin (Dystrophin).
  • FIG 22 shows the germline inheritance of DMD mutant mice (F0 ⁇ F1).
  • Figure 23 is an off-target analysis of the combination of predicted off-target sites of hyA3A-BE4max and DMD-sg3 on the F0 generation.
  • the DNA of the targets EMX1 site1 and Tim3-sg1 shown in Table 2 were artificially synthesized and respectively connected to the BbsI site of the sgRNA expression plasmid U6-sgRNA-EF1 ⁇ -GFP (used to express the sgRNA of the corresponding target) to obtain the recombinant plasmid pE And pT.
  • Target name Sequence SEQ ID No. EMX1 site1 GAGTCCGAGCAGAAGAAGAAGGG 17 Tim3-sg1 TTCTACACCCCAGCCGCCCCAGG 18 VEGFA site2 GACCCCCTCCACCCCGCCTCCGG 19 Lag3-sg2 CGCTACACGGTGCTGAGCGTGGG 20 HEK3 GGCCCAGACTGAGCACGTGATGG twenty one HEK4 GGCACTGCGGCTGGAGGTGGGGG twenty two EMX1-sg2p GACATCGATGTCCTCCCCATTGG twenty three Nme1-sg1 AGGGATCGTCTTTCAAGGCGAGG twenty four
  • each plasmid combination is transfected Set 3 wells to repeat, 2 ⁇ 10 5 cells per well. At the same time, a blank control that does not transfect any plasmid is set.
  • pssDBD-BE4max represents: plasmid pRPA70-A-BE4max, pRPA70-B-BE4max, pRPA70-AB-BE4max, pRPA70-C-BE4max, pRPA32-D-BE4max, pBRCA2-OB2-BE4max, pBRCA2-OB3-BE4max, pKH-
  • the plasmid pCMV-BE4max is used as a negative control.
  • BE4max (Rad51DBD-N-BE4max or Rad51DBD-BE4max) fused with the functional domain of Rad51 single-stranded DNA binding protein has the most significant increase in the editing efficiency of C to T on the target, followed by fusion BE4max of the functional domain of RPA70-C single-stranded DNA binding protein.
  • Rad51 DBD was fused to the other two different positions of BE4max, and Rad51DBD was fused.
  • BE4max (the third to fifth figures from top to bottom in Figure 1), a total of three recombinant plasmids and recombinant plasmid pE or pT were transfected into cells according to the method 1.2 in step 1, and according to 1.3 and The 1.4 method obtains the editing efficiency results (Table 4 and Table 5).
  • Rad51DBD-N-BE4max In BE4max, Rad51 DBD is fused between NLS and rA1, that is, Rad51 DBD is located at the N end of rA1 and spCas9n;
  • Rad51DBD-C-BE4max Fusion Rad51 DBD between spCas9n and UGI in BE4max, that is, Rad51 DBD is located at the C end of rA1 and spCas9n;
  • hyBE4max fusion of Rad51 DBD between rA1 and spCas9n in BE4max.
  • VEGFA site2 Lag3-sg2, HEK3, HEK4, EMX1-sg2p, Nme1-sg1 (sequences shown in Table 2)
  • VEGFA site2 Lag3-sg2, HEK3, HEK4, EMX1-sg2p, Nme1-sg1 (sequences shown in Table 2)
  • EMX1-sg2p Nme1-sg1 (s shown in Table 2)
  • plasmid U6-sgRNA- At the BbsI site of EF1 ⁇ -GFP, recombinant plasmids pV, pL, pH3, pH4, pEP and pN were obtained.
  • the plasmid was sequenced by sanger to ensure that it was completely correct.
  • Rad51-DBD was synthesized according to the coding sequence in Table 1, and then seamlessly cloned and assembled into the plasmid pCMV-A3A-BE4max expressing the protein A3A-BE4max ( Figure 5) between hA3A and spCas9n to construct the expression fusion protein
  • the recombinant plasmid pA of hyA3A-BE4max Figure 5).
  • the target sequences are shown in Table 2, FANCF site1, EGFR-sg5, EGFR
  • the target sequence of -sg21 is shown in Table 6; respectively connected to the BbsI site of the sgRNA expression plasmid pU6-sgRNA-EF1 ⁇ -GFP to obtain recombinant plasmids pB1, pB2, ... pB8 expressing the sgRNA of the corresponding target.
  • Target name Sequence SEQ ID No. FANCF site1 GGAATCCCTTCTGCAGCACCTGG 25 EGFR-sg5 GTGCTGGGCTCCGGTGCGTTCGG 26 EGFR-sg21 CAAAGCAGAAACTCACATCGAGG 27
  • step 1.3 where the identification primers of FANCF site1, EGFR-sg5, and EGFR-sg21 are shown in Table 7, and the remaining expressed identification primers are shown in Table 3.
  • the fusion protein hyA3A-BE4max had significantly improved editing efficiency of a single base C to T at different positions (C3-C15) of each target ( Figure 6).
  • the high activity window of hyA3A-BE4max has expanded from C3-C11 to C3-C15; among them, in C3-C11 far from the PAM region, the editing efficiency of hyA3A-BE4max for a single base C to T is 1.1-2.3 times that of A3A-BE4max.
  • the editing efficiency of hyA3A-BE4max for a single base C to T is 3.1-4.1 times that of A3A-BE4max, that is, at C12-C15 near the PAM region , HyA3A-BE4max improves the editing efficiency of a single base C to T more significantly (Figure 7). And hyA3A-BE4max also maintained low indels (Figure 8).
  • Rad51-DBD was synthesized according to the coding sequence in Table 1, and then seamlessly cloned and assembled into the plasmid pCMV-eA3A-BE4max expressing the protein eA3A-BE4max ( Figure 9) between eA3A and spCas9n to construct the expression fusion protein hyeA3A-BE4max ( Figure 9)
  • the recombinant plasmid pAe The recombinant plasmid pAe.
  • EMX1-sg2p Simultaneously designed and synthesized 11 human endogenous targets: EMX1-sg2p, EMX1 site1, Nme1-sg1.
  • the target sequences are shown in Table 2, and the target sequences of EGFR-sg21 are shown in Table 6, and the rest of the targets The sequence is shown in Table 8, respectively connected to the BbsI site of the sgRNA expression plasmid U6-sgRNA-EF1 ⁇ -GFP to express the sgRNA of the corresponding target to obtain the recombinant plasmid pC1, pC2, ... pC11.
  • Target name Sequence (5 ⁇ -3 ⁇ ) SEQ ID No. CTLA-sg1 CTCCCTCAAGCAGGCCCCGCTGG 28 EGFR-sg5 GTGCTGGGCTCCGGTGCGTTCGG 29 CDK10-sg1 TTCTCGGAGGCTCAGGTGCGTGG 30 EMX1-sg1 GCTCCCATCACATCAACCGGTGG 31 HPRT1-sg6 GCCCTCTGTGTGCTCAAGGGGGG 32 EGFR-sg26 CATGCCCTTCGGCTGCCTCCTGG 33 CCR5-sg1 TAATAATTGATGTCATAGATTGG 34
  • step 1.3 where the identification primers of EMX1-sg2p, EMX1 site1, Nme1-sg1 are shown in Table 3, the identification primers of EGFR-sg21 are shown in Table 7, and the remaining target sequences are shown in Table 9.
  • the fusion protein hyeA3A-BE4max has a significant increase in the editing efficiency of a single base C to T at different positions (C3-C15) of each target site, and the high activity window is expanded from the original C3-C11 To C3-C15, it can specifically target a single base C in the TC motif to achieve C to T conversion (Figure 10); among them, at C3-C11 far away from the PAM region, hyeA3A-BE4max pairs a single base C to T.
  • the editing efficiency of eA3A-BE4max is 1.6-2.8 times that of eA3A-BE4max.
  • the editing efficiency of hyeA3A-BE4max for a single base C to T is 4.5-31.9 times that of eA3A-BE4max, that is, the editing efficiency of hyeA3A-BE4max is 4.5-31.9 times that of eA3A-BE4max.
  • the editing efficiency of single base C to T is improved more obviously ( Figure 11).
  • hyeA3A-BE4max kept low indels ( Figure 12).
  • Method 6 performs deep sequencing and statistical analysis.
  • the construction method of the above recombinant plasmid pC12 is as follows: the sgRNA target sequence of HBG-117G (GGCTATTGGTCAAGGCAAGGCTGG, SEQ ID No. 35) is connected to the BbsI site of the sgRNA expression plasmid U6-sgRNA-EF1 ⁇ -GFP to express the corresponding target Spot the sgRNA to obtain the recombinant plasmid pC12.
  • the identification primers used for the above-mentioned deep sequencing target HBG-117G are as follows:
  • HBG-117G F AGTGAGTACGGTGTGCTGGAATGACTGAATCGGAACAAGGC;
  • HBG-117G R GTTGGATGCTGGATGGCTGGCCTCACTGGATACTCTAAGACT.
  • A3A-BE4max not only targets the -117G>A(C11) mutation in the HBG1/2 promoter region, but also produces -109G>A(C3), -122G>A(C16) "Bystander" mutation; for eA3A-BE4max and hyeA3A-BE4max, eA3A-BE4max accurately edits the G to A transition on -117 (that is, the complementary chain C to T transition) without causing the "bystander" mutation, But the efficiency is very low.
  • hyeA3A-BE4max increases the G to A conversion efficiency by 6.6 times, and there is no detectable "bystander” mutation at -109 and -122.
  • the results show that hyeA3A-BE4max exhibits precise and efficient targeting of -117G in the HBG1/2 promoter region (a summary of the mechanism of action is shown in Figure 13).
  • the hyA3A-BE4max coding sequence was seamlessly cloned to replace BE3 on the backbone vector to obtain the lentiviral vector Lenti-hyA3A-BE4max-P2A-GFP.
  • the hyeA3A-BE4max coding sequence was seamlessly cloned to replace BE3 on the backbone vector to obtain the lentiviral vector Lenti-hyeA3A-BE4max-P2A-GFP.
  • the virus supernatant was collected from HEK293T cell supernatant at 48h after transfection (starting at 0 hours after transfection) and 72h. Centrifuge at 4000g for 10 min at 4°C to remove cell debris, then filter the supernatant with a 0.45 ⁇ m filter in a 40mL ultrafiltration centrifuge tube, add the crude lentivirus extract to the filter cup, and centrifuge at 25000g at 4°C for 2.5 hours. After centrifugation, take out the centrifugal device and separate the filter cup and the filtrate collection cup below.
  • the liquid in the sample collection cup is the virus concentrate (containing the lentivirus Lenti-117G-hyA3A-BE4max-P2A-GFP or the lentivirus Lenti-117G-hyeA3A-BE4max-P2A-GFP). Remove the virus concentrate and store it in virus tubes after aliquoting at -80°C for long-term storage.
  • the HUDEP-2 ( ⁇ G ⁇ ) cells infected with the lentivirus Lenti-117G-hyA3A-BE4max-P2A-GFP or Lenti-117G-hyeA3A-BE4max-P2A-GFP were flow-sorted for GFP-positive cells and cultured until the number of cells was greater than 5 After ⁇ 10 4 , the cells are collected, genomic DNA is extracted, and deep sequencing and analysis are performed according to the method in step 5.1.
  • HUDEP-2 ( ⁇ G ⁇ ) cells infected with the lentivirus Lenti-117G-hyA3A-BE4max-P2A-GFP or Lenti-117G-hyeA3A-BE4max-P2A-GFP were flow-sorted for GFP-positive cells and cultured until the number of cells was greater than 5 ⁇ 10 4 , after about 5-7 days, HUDEP-2 ( ⁇ G ⁇ ) cells were collected for differentiation and expression.
  • the differentiation process is as follows:
  • HUDEP-2 ( ⁇ G ⁇ ) cells were differentiated in erythrocyte-like differentiation medium (IMDM), supplemented with 2% human blood type AB plasma (serum) (Gemini, 100-512), 1% L-glutamine, 2IU/mL heparin, erythropoietin (EPO, 3IU/ml, PeproTech), 330 ⁇ g/mL Holo-human transferrin (Sigma-Aldrich), human stem cell factor (SCF, 50ng/ ml, PeproTech), 2% Pen/Strep (Gibco), 10 ⁇ g/ml recombinant human insulin, differentiation for 8 days.
  • IMDM erythrocyte-like differentiation medium
  • ⁇ -globin expression detection 8 days after differentiation, the cells were harvested and the total mRNA was extracted by phenol-chloroform extraction method.
  • the primers (5’-3’) are as follows:
  • HBG-QPCR-F GGTTATCAATAAGCTCCTAGTCC;
  • HBG-QPCR-R ACAACCAGGAGCCTTCCCA
  • HBB-QPCR-F TGAGGAGAAGTCTGCCGTTAC
  • HBB-QPCR-F ACCACCAGCAGCCTGCCCA.
  • hyA3A-BE4max and hyeA3A-BE4max can significantly increase the level of ⁇ -globin mRNA in HUDEP-2 ( ⁇ G ⁇ ) cells; hyeA3A-BE4max has a positive effect on HUDEP
  • the level of ⁇ -globin mRNA increased in -2 ( ⁇ G ⁇ ) cells was three times that of hyA3A-BE4max ( Figure 17).
  • mice used below are C57/BL6 mice.
  • T7 templates of A3A-BE4max and hyA3A-BE4max were transcribed in vitro to obtain working system mRNA and purified.
  • mRNA containing A3A-BE4max or mRNA containing hyA3A-BE4max a working system mRNA (mRNA containing A3A-BE4max or mRNA containing hyA3A-BE4max) with a total volume of 20 ⁇ L and a final concentration of 100ng/ ⁇ L and sgRNA with a final concentration of 200ng/ ⁇ L (DMD-sg3) mixed solution.
  • the superovulated donor mother mice were killed by carbon dioxide asphyxiation method, and the oviducts were taken out, placed in a petri dish, and preheated M2 medium was added to the petri dish.
  • the target-site DMA-sg3 primer pair F/R (Table 11) was used to obtain the target-containing DNA fragments.
  • high-throughput results among the 10 F0 in the hyA3A-BE4max treatment group, there were 6 homozygous terminating nonsense mutations from CAA to TAA (numbered #BD03, #BD05, # in the bottom figure in Figure 19).
  • BD07, #BD12, #BD15, #BD16 mice) and no termination nonsense mutation from CAA to TAA was found in the A3A-BE4max treatment group F0 ( Figure 19 upper panel).
  • mice in the WT(+/+) and A3A-BE4max treatment groups (such as #AD26)
  • only the mice in the hyA3A-BE4max treatment group caused homozygous C to T at position 10 of the DMA-sg3 target sequence DMD in the 6 F0 generation mice with nonsense mutations (#BD03 in Figure 21) did not get protein expression (Figure 21), which also proved that the DMD animal disease model was successfully constructed.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Public Health (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Environmental Sciences (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Mycology (AREA)
  • Diabetes (AREA)
  • Cell Biology (AREA)
  • Urology & Nephrology (AREA)
  • Rheumatology (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Endocrinology (AREA)
  • Animal Husbandry (AREA)
  • Pathology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
PCT/CN2020/137239 2019-12-18 2020-12-17 一种提高基因编辑效率的融合蛋白及其应用 Ceased WO2021121321A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP20903960.1A EP4079765A4 (en) 2019-12-18 2020-12-17 Fusion protein that improves gene editing efficiency and application thereof
JP2022538379A JP7606176B2 (ja) 2019-12-18 2020-12-17 ゲノム編集効率を向上させる融合タンパク質及びその使用
US17/843,462 US20220364072A1 (en) 2019-12-18 2022-06-17 Fusion protein that improves gene editing efficiency and application thereof

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
CN201911310969.8 2019-12-18
CN201911310969.8A CN112979821B (zh) 2019-12-18 2019-12-18 一种提高基因编辑效率的融合蛋白及其应用
CN201911312544.0A CN112979823B (zh) 2019-12-18 2019-12-18 一种用于治疗和/或预防β血红蛋白病的产品及融合蛋白
CN201911312537.0 2019-12-18
CN201911312544.0 2019-12-18
CN201911312537.0A CN112979822B (zh) 2019-12-18 2019-12-18 一种疾病动物模型的构建方法及融合蛋白

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US17/843,462 Continuation-In-Part US20220364072A1 (en) 2019-12-18 2022-06-17 Fusion protein that improves gene editing efficiency and application thereof

Publications (1)

Publication Number Publication Date
WO2021121321A1 true WO2021121321A1 (zh) 2021-06-24

Family

ID=76477098

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/137239 Ceased WO2021121321A1 (zh) 2019-12-18 2020-12-17 一种提高基因编辑效率的融合蛋白及其应用

Country Status (4)

Country Link
US (1) US20220364072A1 (https=)
EP (1) EP4079765A4 (https=)
JP (1) JP7606176B2 (https=)
WO (1) WO2021121321A1 (https=)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113621634A (zh) * 2021-07-07 2021-11-09 浙江大学杭州国际科创中心 一种增加基因组突变率的碱基编辑系统及碱基编辑方法
EP4400586A4 (en) * 2021-09-07 2025-04-30 National University Corporation Chiba University Composition for modifying nucleic acid sequence, and method for modifying target site of nucleic acid sequence

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102616916B1 (ko) * 2021-05-11 2023-12-21 주식회사 애이마 발암성 돌연변이 유전자 검출용 가이드 rna 내지 이의 용도

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105647968A (zh) * 2016-02-02 2016-06-08 浙江大学 一种CRISPR/Cas9工作效率快速测试系统及其应用
WO2017142923A1 (en) * 2016-02-16 2017-08-24 Emendobio Inc. Compositions and methods for promoting homology directed repair mediated gene editing
CN108350449A (zh) * 2015-08-28 2018-07-31 通用医疗公司 工程化的CRISPR-Cas9核酸酶
CN108588182A (zh) * 2018-04-13 2018-09-28 中国科学院深圳先进技术研究院 基于crispr-链取代的等温扩增及检测技术
CN108707635A (zh) * 2018-05-29 2018-10-26 华东师范大学 用于核苷酸序列修饰的组合物、方法与应用
CN109880851A (zh) * 2019-03-28 2019-06-14 西北农林科技大学 用于富集CRISPR/Cas9介导的同源重组修复细胞的筛选报告载体及筛选方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010132092A2 (en) * 2009-05-12 2010-11-18 The Scripps Research Institute Cytidine deaminase fusions and related methods
ITFI20120173A1 (it) * 2012-08-30 2014-03-01 Selex Galileo Spa "sistema di visione con telecamera e sistema di limitazione delle sollecitazioni"
US20150165054A1 (en) * 2013-12-12 2015-06-18 President And Fellows Of Harvard College Methods for correcting caspase-9 point mutations
KR20240116572A (ko) * 2017-03-23 2024-07-29 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 핵산 프로그램가능한 dna 결합 단백질을 포함하는 핵염기 편집제
EP3622070A2 (en) * 2017-05-10 2020-03-18 Editas Medicine, Inc. Crispr/rna-guided nuclease systems and methods

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108350449A (zh) * 2015-08-28 2018-07-31 通用医疗公司 工程化的CRISPR-Cas9核酸酶
CN105647968A (zh) * 2016-02-02 2016-06-08 浙江大学 一种CRISPR/Cas9工作效率快速测试系统及其应用
WO2017142923A1 (en) * 2016-02-16 2017-08-24 Emendobio Inc. Compositions and methods for promoting homology directed repair mediated gene editing
CN108588182A (zh) * 2018-04-13 2018-09-28 中国科学院深圳先进技术研究院 基于crispr-链取代的等温扩增及检测技术
CN108707635A (zh) * 2018-05-29 2018-10-26 华东师范大学 用于核苷酸序列修饰的组合物、方法与应用
CN109880851A (zh) * 2019-03-28 2019-06-14 西北农林科技大学 用于富集CRISPR/Cas9介导的同源重组修复细胞的筛选报告载体及筛选方法

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
CHUAI, G. ET AL.: "DeepCRISPR: Optimized CRISPR Guide RNA Design by Deep Learning", GENOME BIOLOGY, vol. 19, 31 December 2018 (2018-12-31), XP055716006, DOI: 10.1186/s13059-018-1459-4 *
GUOHUI CHUAI, HANHUI MA, JIFANG YAN, MING CHEN, NANFANG HONG, DONGYU XUE, CHI ZHOU, CHENYU ZHU, KE CHEN, BIN DUAN, FENG GU, SHENG : "DeepCRISPR: optimized CRISPR guide RNA design by deep learning", GENOME BIOLOGY, vol. 19, no. 1, 1 December 2018 (2018-12-01), XP055716006, DOI: 10.1186/s13059-018-1459-4 *
HAVLICEK STEVEN, SHEN YANG, ALPAGU YUNUS, BRUNTRAEGER MICHAELA B., ZUFIR NURDIANA B.M., PHUAH ZHI YI, FU ZHIYAN, DUNN NORRIS R., S: "Re-engineered RNA-Guided FokI-Nucleases for Improved Genome Editing in Human Cells", MOLECULAR THERAPY, vol. 25, no. 2, 1 February 2017 (2017-02-01), US, pages 342 - 355, XP055822926, ISSN: 1525-0016, DOI: 10.1016/j.ymthe.2016.11.007 *
JASON M GEHRKE, OLIVER CERVANTES, M KENDELL CLEMENT, YUXUAN WU, JING ZENG, DANIEL E BAUER, LUCA PINELLO, J KEITH JOUNG: "An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities", NATURE BIOTECHNOLOGY, vol. 36, no. 10, 1 November 2018 (2018-11-01), New York, pages 977 - 982, XP055632872, ISSN: 1087-0156, DOI: 10.1038/nbt.4199 *
KOBLAN LUKE W; DOMAN JORDAN L; WILSON CHRISTOPHER; LEVY JONATHAN M; TAY TRISTAN; NEWBY GREGORY A; MAIANTI JUAN PABLO; RAGURAM ADIT: "Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction", NATURE BIOTECHNOLOGY, vol. 36, no. 9, 1 October 2018 (2018-10-01), New York, pages 843 - 846, XP036929657, ISSN: 1087-0156, DOI: 10.1038/nbt.4172 *
See also references of EP4079765A4
ZHANG XIAOHUI; CHEN LIANG; ZHU BIYUN; WANG LIREN; CHEN CAIYU; HONG MENGJIA; HUANG YIFAN; LI HUIYING; HAN HONGHUI; CAI BAILIAN; YU : "Increasing the efficiency and targeting range of cytidine base editors through fusion of a single-stranded DNA-binding protein domain", NATURE CELL BIOLOGY, vol. 22, no. 6, 11 May 2020 (2020-05-11), GB, pages 740 - 750, XP037159237, ISSN: 1465-7392, DOI: 10.1038/s41556-020-0518-8 *
ZHANG, XIAOHUI: "The Optimization and Application of Base Editing Technology", CHINA DOCTORAL DISSERTATIONS FULL-TEXT DATABASE (BASIC SCIENCES), no. 8, 15 August 2020 (2020-08-15), pages 1 - 167, XP055823124 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113621634A (zh) * 2021-07-07 2021-11-09 浙江大学杭州国际科创中心 一种增加基因组突变率的碱基编辑系统及碱基编辑方法
CN113621634B (zh) * 2021-07-07 2023-09-15 浙江大学杭州国际科创中心 一种增加基因组突变率的碱基编辑系统及碱基编辑方法
EP4400586A4 (en) * 2021-09-07 2025-04-30 National University Corporation Chiba University Composition for modifying nucleic acid sequence, and method for modifying target site of nucleic acid sequence

Also Published As

Publication number Publication date
JP7606176B2 (ja) 2024-12-25
EP4079765A1 (en) 2022-10-26
JP2023507034A (ja) 2023-02-20
US20220364072A1 (en) 2022-11-17
EP4079765A4 (en) 2024-02-14

Similar Documents

Publication Publication Date Title
CN106244557B (zh) 定点突变ApoE基因与LDLR基因的方法
CN105907758B (zh) CRISPR-Cas9引导序列及其引物、转基因表达载体及其构建方法
CN108285906B (zh) 一种定点整合外源dna转基因猪的构建方法
CN107619837A (zh) 利用Cas9切割核酸酶介导Ipr1定点插入获取转基因牛胎儿成纤维细胞的方法
US20220364072A1 (en) Fusion protein that improves gene editing efficiency and application thereof
CN105492609A (zh) CRISPR-Cas9特异性敲除猪GGTA1基因的方法及用于特异性靶向GGTA1基因的sgRNA
CN105518135A (zh) CRISPR-Cas9特异性敲除猪CMAH基因的方法及用于特异性靶向CMAH基因的sgRNA
CN106414740A (zh) CRISPR‑Cas9特异性敲除猪SLA‑3基因的方法及用于特异性靶向SLA‑3基因的sgRNA
CN105492608A (zh) CRISPR-Cas9特异性敲除猪PDX1基因的方法及用于特异性靶向PDX1基因的sgRNA
CN105518134A (zh) CRISPR-Cas9特异性敲除猪SLA-2基因的方法及用于特异性靶向SLA-2基因的sgRNA
CN109750035B (zh) 靶向并引导Cas9蛋白高效切割TCR及B2M基因座的sgRNA
CN108642054A (zh) 降低免疫原性sgRNA、低免疫原性敷料及其制备方法
WO2019154437A1 (zh) CRISPR/Cas9载体组合及其在基因敲除中的应用
CN102296073B (zh) 锌指核酸酶定点敲除Myostatin基因特异靶位点
CN104212837B (zh) 表达人血清白蛋白的慢病毒载体及其构建方法
CN103805606B (zh) 一对特异识别绵羊DKK1基因的sgRNA及其编码DNA和应用
CN106282230B (zh) 定点突变ldlr基因的方法
WO2024183423A1 (zh) CRISPR/Cas9-gRNA打靶质粒、供体质粒以及永生化小鼠细胞系的制备方法
WO2022012512A1 (zh) 敲除猪异种抗原的基因的gRNA及其应用
CN106244556B (zh) 定点突变ApoE基因的方法
CN113201499A (zh) Hbb基因cd17突变细胞及其制备方法与应用
CN115948465B (zh) 猪hat1基因修饰系统及应用
CN115678914A (zh) 一种由精液介导的转基因鸡输卵管生物反应器的制备方法
CN112979822A (zh) 一种疾病动物模型的构建方法及融合蛋白
CN107018955A (zh) 一种抗猪圆环病毒2型的转基因猪

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20903960

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2022538379

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020903960

Country of ref document: EP

Effective date: 20220718