WO2023216415A1 - 基于双分子脱氨酶互补的碱基编辑系统及其应用 - Google Patents
基于双分子脱氨酶互补的碱基编辑系统及其应用 Download PDFInfo
- Publication number
- WO2023216415A1 WO2023216415A1 PCT/CN2022/106402 CN2022106402W WO2023216415A1 WO 2023216415 A1 WO2023216415 A1 WO 2023216415A1 CN 2022106402 W CN2022106402 W CN 2022106402W WO 2023216415 A1 WO2023216415 A1 WO 2023216415A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- base editing
- fusion protein
- split
- base
- amino acid
- Prior art date
Links
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 100
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 100
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims abstract description 72
- 108020005004 Guide RNA Proteins 0.000 claims abstract description 56
- 229940104302 cytosine Drugs 0.000 claims abstract description 36
- 229930024421 Adenine Natural products 0.000 claims abstract description 33
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims abstract description 33
- 229960000643 adenine Drugs 0.000 claims abstract description 33
- 241000282414 Homo sapiens Species 0.000 claims abstract description 31
- 230000001419 dependent effect Effects 0.000 claims abstract description 16
- 230000000295 complement effect Effects 0.000 claims abstract description 15
- 241001465754 Metazoa Species 0.000 claims abstract description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 102
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 73
- 239000012634 fragment Substances 0.000 claims description 72
- 229920001184 polypeptide Polymers 0.000 claims description 71
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 71
- 240000007594 Oryza sativa Species 0.000 claims description 51
- 235000007164 Oryza sativa Nutrition 0.000 claims description 46
- 235000009566 rice Nutrition 0.000 claims description 45
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 44
- 210000004027 cell Anatomy 0.000 claims description 36
- 241000196324 Embryophyta Species 0.000 claims description 33
- 230000014509 gene expression Effects 0.000 claims description 30
- 241000219194 Arabidopsis Species 0.000 claims description 27
- 108010075254 C-Peptide Proteins 0.000 claims description 24
- 241000219195 Arabidopsis thaliana Species 0.000 claims description 22
- 239000002773 nucleotide Substances 0.000 claims description 21
- 125000003729 nucleotide group Chemical group 0.000 claims description 21
- 102100026846 Cytidine deaminase Human genes 0.000 claims description 18
- 108010031325 Cytidine deaminase Proteins 0.000 claims description 18
- 101710169336 5'-deoxyadenosine deaminase Proteins 0.000 claims description 16
- 102000055025 Adenosine deaminases Human genes 0.000 claims description 16
- 108020004414 DNA Proteins 0.000 claims description 14
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims description 12
- 101000964378 Homo sapiens DNA dC->dU-editing enzyme APOBEC-3A Proteins 0.000 claims description 9
- 101000964385 Homo sapiens DNA dC->dU-editing enzyme APOBEC-3B Proteins 0.000 claims description 9
- 210000004899 c-terminal region Anatomy 0.000 claims description 9
- 102100040263 DNA dC->dU-editing enzyme APOBEC-3A Human genes 0.000 claims description 8
- 102100040262 DNA dC->dU-editing enzyme APOBEC-3B Human genes 0.000 claims description 8
- 102100040261 DNA dC->dU-editing enzyme APOBEC-3C Human genes 0.000 claims description 8
- 102100040264 DNA dC->dU-editing enzyme APOBEC-3D Human genes 0.000 claims description 8
- 102100040266 DNA dC->dU-editing enzyme APOBEC-3F Human genes 0.000 claims description 8
- 102100038076 DNA dC->dU-editing enzyme APOBEC-3G Human genes 0.000 claims description 8
- 102100038050 DNA dC->dU-editing enzyme APOBEC-3H Human genes 0.000 claims description 8
- 101710082737 DNA dC->dU-editing enzyme APOBEC-3H Proteins 0.000 claims description 8
- 101000964383 Homo sapiens DNA dC->dU-editing enzyme APOBEC-3C Proteins 0.000 claims description 8
- 101000964382 Homo sapiens DNA dC->dU-editing enzyme APOBEC-3D Proteins 0.000 claims description 8
- 101000964377 Homo sapiens DNA dC->dU-editing enzyme APOBEC-3F Proteins 0.000 claims description 8
- 150000001413 amino acids Chemical class 0.000 claims description 8
- 240000008042 Zea mays Species 0.000 claims description 7
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 7
- 108010029988 AICDA (activation-induced cytidine deaminase) Proteins 0.000 claims description 6
- 241000233866 Fungi Species 0.000 claims description 6
- 102100022433 Single-stranded DNA cytosine deaminase Human genes 0.000 claims description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 6
- 235000005822 corn Nutrition 0.000 claims description 6
- 229940035893 uracil Drugs 0.000 claims description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 5
- 241000700159 Rattus Species 0.000 claims description 5
- 230000003612 virological effect Effects 0.000 claims description 5
- 108010079649 APOBEC-1 Deaminase Proteins 0.000 claims description 4
- 108010004483 APOBEC-3G Deaminase Proteins 0.000 claims description 4
- 102100040397 C->U-editing enzyme APOBEC-1 Human genes 0.000 claims description 4
- 102100040399 C->U-editing enzyme APOBEC-2 Human genes 0.000 claims description 4
- 101000964322 Homo sapiens C->U-editing enzyme APOBEC-2 Proteins 0.000 claims description 4
- 101000800426 Homo sapiens Putative C->U-editing enzyme APOBEC-4 Proteins 0.000 claims description 4
- 102100033091 Putative C->U-editing enzyme APOBEC-4 Human genes 0.000 claims description 4
- 244000062793 Sorghum vulgare Species 0.000 claims description 4
- 230000001105 regulatory effect Effects 0.000 claims description 4
- 101150094690 GAL1 gene Proteins 0.000 claims description 3
- 102100028501 Galanin peptides Human genes 0.000 claims description 3
- 229940122069 Glycosidase inhibitor Drugs 0.000 claims description 3
- 101100121078 Homo sapiens GAL gene Proteins 0.000 claims description 3
- 235000021307 Triticum Nutrition 0.000 claims description 3
- 239000003316 glycosidase inhibitor Substances 0.000 claims description 3
- 230000030648 nucleus localization Effects 0.000 claims description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 2
- 241000283690 Bos taurus Species 0.000 claims description 2
- 241000282693 Cercopithecidae Species 0.000 claims description 2
- 229920000742 Cotton Polymers 0.000 claims description 2
- 102000053602 DNA Human genes 0.000 claims description 2
- 241000588724 Escherichia coli Species 0.000 claims description 2
- 241000282326 Felis catus Species 0.000 claims description 2
- 244000068988 Glycine max Species 0.000 claims description 2
- 235000010469 Glycine max Nutrition 0.000 claims description 2
- 244000299507 Gossypium hirsutum Species 0.000 claims description 2
- 235000003222 Helianthus annuus Nutrition 0.000 claims description 2
- 244000020551 Helianthus annuus Species 0.000 claims description 2
- 101000755690 Homo sapiens Single-stranded DNA cytosine deaminase Proteins 0.000 claims description 2
- 240000005979 Hordeum vulgare Species 0.000 claims description 2
- 235000007340 Hordeum vulgare Nutrition 0.000 claims description 2
- 235000007688 Lycopersicon esculentum Nutrition 0.000 claims description 2
- 240000003183 Manihot esculenta Species 0.000 claims description 2
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 claims description 2
- 240000004658 Medicago sativa Species 0.000 claims description 2
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 claims description 2
- 244000061176 Nicotiana tabacum Species 0.000 claims description 2
- 235000002637 Nicotiana tabacum Nutrition 0.000 claims description 2
- 241001494479 Pecora Species 0.000 claims description 2
- 240000000111 Saccharum officinarum Species 0.000 claims description 2
- 235000007201 Saccharum officinarum Nutrition 0.000 claims description 2
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 2
- 240000003768 Solanum lycopersicum Species 0.000 claims description 2
- 244000061456 Solanum tuberosum Species 0.000 claims description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 2
- 235000011684 Sorghum saccharatum Nutrition 0.000 claims description 2
- 235000003869 genetically modified organism Nutrition 0.000 claims description 2
- 235000019713 millet Nutrition 0.000 claims description 2
- 241000699666 Mus <mouse, genus> Species 0.000 claims 1
- 241000009328 Perro Species 0.000 claims 1
- 241000282898 Sus scrofa Species 0.000 claims 1
- 244000098338 Triticum aestivum Species 0.000 claims 1
- 229930182470 glycoside Natural products 0.000 claims 1
- 150000002338 glycosides Chemical class 0.000 claims 1
- 108091033409 CRISPR Proteins 0.000 abstract description 21
- 208000026350 Inborn Genetic disease Diseases 0.000 abstract description 5
- 208000016361 genetic disease Diseases 0.000 abstract description 5
- 230000006872 improvement Effects 0.000 abstract description 3
- 238000012214 genetic breeding Methods 0.000 abstract description 2
- 239000013598 vector Substances 0.000 description 46
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 40
- 239000013604 expression vector Substances 0.000 description 39
- 230000002068 genetic effect Effects 0.000 description 26
- 238000000034 method Methods 0.000 description 26
- 108090000623 proteins and genes Proteins 0.000 description 26
- 230000009466 transformation Effects 0.000 description 26
- 238000010276 construction Methods 0.000 description 25
- 210000001938 protoplast Anatomy 0.000 description 25
- 230000009261 transgenic effect Effects 0.000 description 23
- 239000000243 solution Substances 0.000 description 21
- 238000012350 deep sequencing Methods 0.000 description 14
- 238000004458 analytical method Methods 0.000 description 13
- 230000037429 base substitution Effects 0.000 description 12
- 235000001014 amino acid Nutrition 0.000 description 11
- 238000010586 diagram Methods 0.000 description 11
- 239000000463 material Substances 0.000 description 11
- 239000013612 plasmid Substances 0.000 description 11
- 230000000694 effects Effects 0.000 description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 8
- 210000003292 kidney cell Anatomy 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 238000012163 sequencing technique Methods 0.000 description 8
- 238000006467 substitution reaction Methods 0.000 description 8
- 239000004009 herbicide Substances 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 238000001890 transfection Methods 0.000 description 7
- 101000910035 Streptococcus pyogenes serotype M1 CRISPR-associated endonuclease Cas9/Csn1 Proteins 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 238000001976 enzyme digestion Methods 0.000 description 6
- 150000007523 nucleic acids Chemical group 0.000 description 6
- 108091033319 polynucleotide Proteins 0.000 description 6
- 102000040430 polynucleotide Human genes 0.000 description 6
- 239000002157 polynucleotide Substances 0.000 description 6
- 235000018102 proteins Nutrition 0.000 description 6
- 230000008685 targeting Effects 0.000 description 6
- 210000005253 yeast cell Anatomy 0.000 description 6
- 101150001232 ALS gene Proteins 0.000 description 5
- 230000009437 off-target effect Effects 0.000 description 5
- 238000007480 sanger sequencing Methods 0.000 description 5
- 230000010474 transient expression Effects 0.000 description 5
- CAAMSDWKXXPUJR-UHFFFAOYSA-N 3,5-dihydro-4H-imidazol-4-one Chemical compound O=C1CNC=N1 CAAMSDWKXXPUJR-UHFFFAOYSA-N 0.000 description 4
- 108091093088 Amplicon Proteins 0.000 description 4
- 101710172824 CRISPR-associated endonuclease Cas9 Proteins 0.000 description 4
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 4
- 229930195725 Mannitol Natural products 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 239000000594 mannitol Substances 0.000 description 4
- 235000010355 mannitol Nutrition 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 4
- 101001055189 Arabidopsis thaliana Acetolactate synthase, chloroplastic Proteins 0.000 description 3
- 241000206602 Eukaryota Species 0.000 description 3
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 3
- 101150063416 add gene Proteins 0.000 description 3
- 230000009418 agronomic effect Effects 0.000 description 3
- 235000004279 alanine Nutrition 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000009615 deamination Effects 0.000 description 3
- 238000006481 deamination reaction Methods 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 230000007071 enzymatic hydrolysis Effects 0.000 description 3
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000010362 genome editing Methods 0.000 description 3
- 230000002363 herbicidal effect Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
- 238000011426 transformation method Methods 0.000 description 3
- 241000589158 Agrobacterium Species 0.000 description 2
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 2
- 101000766208 Arabidopsis thaliana BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 101710097330 Bidirectional sugar transporter SWEET11 Proteins 0.000 description 2
- 101000909256 Caldicellulosiruptor bescii (strain ATCC BAA-1888 / DSM 6725 / Z-1320) DNA polymerase I Proteins 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 2
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 101000902592 Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1) DNA polymerase Proteins 0.000 description 2
- 241000793189 Saccharomyces cerevisiae BY4741 Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 108091027544 Subgenomic mRNA Proteins 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- 241000209140 Triticum Species 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 229930182830 galactose Natural products 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 238000012165 high-throughput sequencing Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 229940113082 thymine Drugs 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- DIGQNXIGRZPYDK-WKSCXVIASA-N (2R)-6-amino-2-[[2-[[(2S)-2-[[2-[[(2R)-2-[[(2S)-2-[[(2R,3S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2R)-2-[[2-[[2-[[2-[(2-amino-1-hydroxyethylidene)amino]-3-carboxy-1-hydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1,5-dihydroxy-5-iminopentylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]hexanoic acid Chemical compound C[C@@H]([C@@H](C(=N[C@@H](CS)C(=N[C@@H](C)C(=N[C@@H](CO)C(=NCC(=N[C@@H](CCC(=N)O)C(=NC(CS)C(=N[C@H]([C@H](C)O)C(=N[C@H](CS)C(=N[C@H](CO)C(=NCC(=N[C@H](CS)C(=NCC(=N[C@H](CCCCN)C(=O)O)O)O)O)O)O)O)O)O)O)O)O)O)O)N=C([C@H](CS)N=C([C@H](CO)N=C([C@H](CO)N=C([C@H](C)N=C(CN=C([C@H](CO)N=C([C@H](CS)N=C(CN=C(C(CS)N=C(C(CC(=O)O)N=C(CN)O)O)O)O)O)O)O)O)O)O)O)O DIGQNXIGRZPYDK-WKSCXVIASA-N 0.000 description 1
- FQVLRGLGWNWPSS-BXBUPLCLSA-N (4r,7s,10s,13s,16r)-16-acetamido-13-(1h-imidazol-5-ylmethyl)-10-methyl-6,9,12,15-tetraoxo-7-propan-2-yl-1,2-dithia-5,8,11,14-tetrazacycloheptadecane-4-carboxamide Chemical compound N1C(=O)[C@@H](NC(C)=O)CSSC[C@@H](C(N)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C)NC(=O)[C@@H]1CC1=CN=CN1 FQVLRGLGWNWPSS-BXBUPLCLSA-N 0.000 description 1
- HBEMYXWYRXKRQI-UHFFFAOYSA-N 3-(8-methoxyoctoxy)propyl-methyl-bis(trimethylsilyloxy)silane Chemical compound COCCCCCCCCOCCC[Si](C)(O[Si](C)(C)C)O[Si](C)(C)C HBEMYXWYRXKRQI-UHFFFAOYSA-N 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 102100034035 Alcohol dehydrogenase 1A Human genes 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 108010011485 Aspartame Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 240000002791 Brassica napus Species 0.000 description 1
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000701489 Cauliflower mosaic virus Species 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 102220605874 Cytosolic arginine sensor for mTORC1 subunit 2_D10A_mutation Human genes 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 101710177611 DNA polymerase II large subunit Proteins 0.000 description 1
- 101710184669 DNA polymerase II small subunit Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 101150100333 FLS2 gene Proteins 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 101000892220 Geobacillus thermodenitrificans (strain NG80-2) Long-chain-alcohol dehydrogenase 1 Proteins 0.000 description 1
- 229940113491 Glycosylase inhibitor Drugs 0.000 description 1
- 101000780443 Homo sapiens Alcohol dehydrogenase 1A Proteins 0.000 description 1
- GRRNUXAQVGOGFE-UHFFFAOYSA-N Hygromycin-B Natural products OC1C(NC)CC(N)C(O)C1OC1C2OC3(C(C(O)C(O)C(C(N)CO)O3)O)OC2C(O)C(CO)O1 GRRNUXAQVGOGFE-UHFFFAOYSA-N 0.000 description 1
- 241000209510 Liliopsida Species 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 102000003792 Metallothionein Human genes 0.000 description 1
- 108090000157 Metallothionein Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 101000630792 Oryza sativa subsp. japonica Bidirectional sugar transporter SWEET13 Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 1
- 108010059820 Polygalacturonase Proteins 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 244000184734 Pyrus japonica Species 0.000 description 1
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 241000714474 Rous sarcoma virus Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 1
- OHVGNSMTLSKTGN-BTVCFUMJSA-N [C].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O Chemical compound [C].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O OHVGNSMTLSKTGN-BTVCFUMJSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 239000000605 aspartame Substances 0.000 description 1
- IAOZJIPTCAWIRG-QWRGUYRKSA-N aspartame Chemical group OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)OC)CC1=CC=CC=C1 IAOZJIPTCAWIRG-QWRGUYRKSA-N 0.000 description 1
- 235000010357 aspartame Nutrition 0.000 description 1
- 229960003438 aspartame Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 238000004422 calculation algorithm Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 244000038559 crop plants Species 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000005782 double-strand break Effects 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 108010093305 exopolygalacturonase Proteins 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000006481 glucose medium Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- GRRNUXAQVGOGFE-NZSRVPFOSA-N hygromycin B Chemical compound O[C@@H]1[C@@H](NC)C[C@@H](N)[C@H](O)[C@H]1O[C@H]1[C@H]2O[C@@]3([C@@H]([C@@H](O)[C@@H](O)[C@@H](C(N)CO)O3)O)O[C@H]2[C@@H](O)[C@@H](CO)O1 GRRNUXAQVGOGFE-NZSRVPFOSA-N 0.000 description 1
- 229940097277 hygromycin b Drugs 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007914 intraventricular administration Methods 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 description 1
- 235000009973 maize Nutrition 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000012223 nuclear import Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 1
- 108010054624 red fluorescent protein Proteins 0.000 description 1
- 230000008263 repair mechanism Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
Images
Classifications
-
- 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
- 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
- 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)
-
- 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)
-
- 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
- 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 a base editing system based on bimolecular deaminase complementation and its application, and belongs to the technical field of genetic engineering.
- Base editing technology derived from CRISPR/Cas9 gene editing technology has the characteristics of not producing DNA double-strand breaks and can accurately achieve single base replacement at the target site, which greatly improves the accuracy and accuracy of gene editing. Because of its safety, it is widely used in fields such as gene function research, crop genetic improvement and clinical treatment of human genetic diseases [3] .
- the base editing system mainly consists of base editors (BEs) and guide RNA (single-guide RNA, sgRNA).
- BE3 combines rat cytidine deaminase rAPOBEC1, bacteriophage-derived Uracil glycosylase inhibitor (UGI) and Cas9 nickase (nCas9-D10A). ) fusion to obtain [4] .
- UMI bacteriophage-derived Uracil glycosylase inhibitor
- nCas9-D10A Cas9 nickase
- the purpose of the present invention is to provide a novel base editing system based on bimolecular deaminase complementation and its application.
- the base editing system based on bimolecular deaminase complementation proposed by the invention can greatly reduce the cost of traditional base editing.
- the system solves the genome-wide random off-target problem while still maintaining efficient on-target editing, which has broad application prospects.
- the technical solution adopted by the present invention is: a bimolecular deaminase complementary base editing system, the base editing system includes at least one of the following 1) to 5):
- Base editing fusion protein A Base editing fusion protein A, base editing fusion protein B and guide RNA
- An expression construct comprising the nucleotide sequences encoding base editing fusion protein A and base editing fusion protein B, and guide RNA;
- Base editing fusion protein A Base editing fusion protein B, and an expression construct containing a nucleotide sequence encoding a guide RNA
- An expression construct comprising a nucleotide sequence encoding base editing fusion protein A and base editing fusion protein B and a nucleotide sequence encoding guide RNA;
- the base editing fusion protein A includes the first nCas9 polypeptide fragment, the flexible connecting peptide and the first nucleobase deaminase polypeptide fragment in sequence from the N-terminus to the C-terminus; the base editing fusion protein B includes from the N-terminus
- the C-terminal includes a second nucleobase deaminase polypeptide fragment, a flexible linker peptide and a second nCas9 polypeptide fragment in sequence; the first nucleobase deaminase polypeptide fragment and the second nucleobase deaminase polypeptide fragment are selected from from the same nucleobase deaminase.
- expression construct refers to a vector, such as a recombinant vector, suitable for expressing the nucleotide sequence of interest in an organism. "Expression” means that a functional product can be produced.
- expression of a nucleotide sequence may refer to transcription of the nucleotide sequence (eg, transcription to produce mRNA or functional RNA) and/or translation of the RNA into a precursor or mature protein.
- the "expression construct” of the present invention can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or an RNA capable of translation (such as mRNA).
- the "expression construct” of the present invention may include regulatory sequences and nucleotide sequences of interest from the same source but in a different arrangement than the natural arrangement, as well as regulatory sequences and nucleotide sequences of interest from different sources.
- the guide RNA can form a protein nucleic acid complex (RNP) with the base editing fusion protein A and the base editing fusion protein B, and guide the RNP to target the target DNA sequence, resulting in nucleotide substitution of one or more cytosine bases to thymine bases, or one or more adenine (A) bases to guanine (G) bases in the target sequence.
- RNP protein nucleic acid complex
- the first nucleobase deaminase polypeptide fragment nor the second nucleobase deaminase polypeptide fragment contains a complete deaminase sequence, nor does it have base deamination activity, which is the key to reducing off-target effects of this system. .
- the first and second nucleobase deaminase are usually complementary, that is, they can be fused into a nucleobase deaminase with complete deamination activity. Aminase.
- the amino acid sequence of the first nCas9 polypeptide fragment is as shown in SEQ ID NO: 1; the amino acid sequence of the second nCas9 polypeptide fragment is as shown in SEQ ID NO: 2 Show.
- the flexible connecting peptide includes a 32aa connecting peptide with an amino acid sequence as shown in SEQ ID NO: 3. Research shows that the flexible connecting peptide also includes XTEN, PR, GGGGS, PRGGSGG, ARGGSGG, GS, GAP, (GGGGS) ⁇ 3, GGS and (GGS) ⁇ 7.
- the flexible connecting peptide can also be a non-functional amino acid sequence of 1 to 50 or more amino acids in length and without secondary or higher structures.
- the N-terminal or C-terminal of the base editing fusion protein A and the base editing fusion protein B is fused with at least one nuclear localization sequence.
- the nuclear localization sequence can usually interact with nuclear import carriers, thereby enabling target proteins to be transported into the nucleus.
- NLS consists of one or more short sequences of positively charged lysine or arginine exposed on the protein surface, but other types of NLS are also known.
- Non-limiting examples of NLS include amino acid sequences such as PKKKRKV or KRPAATKKAGQAKKKK.
- the N-terminus of the base editing fusion protein A of the present invention contains 1 copy of NLS (amino acid sequence is PKKKRKV), and the C-terminus of the base editing fusion protein B contains 1 copy of NLS.
- NLS amino acid sequence KRPAATKKAGQAKKKK
- 2 copies of NLS amino acid sequence PKKKRKV and KRPAATKKAGQAKKKK.
- the base editing system is a cytosine base editor system; the nucleobase deaminase is a cytidine deaminase; using this system, the system can be used in the wizard Under the guidance of RNA, one or more C bases in the target sequence are replaced with T bases.
- the cytidine deaminase is selected from: APOBEC3A (A3A), APOBEC3B (A3B), APOBEC3C (A3C), APOBEC3D (A3D), APOBEC3F (A3F), APOBEC3G(A3G), APOBEC3H(A3H), APOBEC1(A1), APOBEC3(A3), APOBEC2(A2), APOBEC4(A4) and AICDA(AID) or by mutating APOBEC3A(A3A), APOBEC3B( A3B), APOBEC3C(A3C), APOBEC3D(A3D), APOBEC3F(A3F), APOBEC3G(A3G), APOBEC3H(A3H), APOBEC1(A1), APOBEC3(A3), APOBEC2(A2), APOBEC4(A4) and A
- the cytidine deaminase is selected from the group consisting of human AID highly active variant AID10 (Chinese Patent: 202010285948.1), rat-derived rA1, human-derived hA3A or human source of hA3B.
- the first nucleobase deaminase polypeptide fragment of the cytosine base editing fusion protein A includes any amino acid sequence shown in SEQ ID NO: 4-8 ;
- the second nucleobase deaminase polypeptide fragment of the cytosine base editing fusion protein B includes any amino acid sequence shown in SEQ ID NO: 9-13.
- the C-terminus of the cytosine base editing fusion protein B is fused with at least one uracil glycosidase inhibitor (UGI), and the amino acid sequence of the UGI is such as SEQ Shown as ID NO:14
- the cytosine base editing system includes a combination of base editing fusion protein A and base editing fusion protein B described in any one of 1) to 5) below. :
- Base editing fusion protein A with the amino acid sequence shown in SEQ ID NO:15: Split-AID10-N
- base editing fusion protein B with the amino acid sequence shown in SEQ ID NO:16: Split-AID10-C ;
- Base editing fusion protein A with the amino acid sequence shown in SEQ ID NO:17: Split-AID10-N5, and base editing fusion protein B with the amino acid sequence shown in SEQ ID NO:18: Split-AID10-C4 ;
- Base editing fusion protein A with the amino acid sequence shown in SEQ ID NO:19: Split-BE3-N
- base editing fusion protein B with the amino acid sequence shown in SEQ ID NO:20: Split-BE3-C ;
- Base editing fusion protein A with the amino acid sequence shown in SEQ ID NO:21: Split-A3A-N
- base editing fusion protein B with the amino acid sequence shown in SEQ ID NO:22: Split-A3A-C ;
- Base editing fusion protein A with the amino acid sequence shown in SEQ ID NO:23: Split-A3B-N
- base editing fusion protein B with the amino acid sequence shown in SEQ ID NO:24: Split-A3B-C .
- the system is based on a bimolecular, cytosine base editing system "Split-AID10" complementary to cytidine deaminase AID10.
- the base editing fusion protein A consists of an NLS polypeptide fragment, a first nCas9 polypeptide fragment (the amino acid sequence is shown in SEQ ID NO: 1), a 32aa connecting peptide (the amino acid sequence is shown in SEQ ID NO: 3) and the first
- the nucleobase polypeptide fragment AID10-N (the amino acid sequence is shown in SEQ ID NO: 4) is sequentially fused from the N end to the C end.
- the base editing fusion protein B consists of the second nucleobase polypeptide fragment AID10-C (the amino acid sequence is as shown in SEQ ID NO: 9), the 32aa connecting peptide, and the second nCas9 polypeptide fragment (the amino acid sequence is as shown in SEQ ID NO:2), 1 molecule of UGI (the amino acid sequence is as shown in SEQ ID NO:14) and 1 molecule of NLS are sequentially fused from the N-terminus to the C-terminus, and its amino acid sequence is as shown in SEQ ID NO:16. It is called It is "Split-AID10-C" (shown in Figure 2).
- the system is based on the bimolecular, cytidine deaminase AID10 complementary cytosine base editing system "Split-AID10-N5-C4".
- the base editing fusion protein A consists of an NLS polypeptide fragment, a first nCas9 polypeptide fragment, a 32aa connecting peptide and a first nucleobase polypeptide fragment AID10-N5 (the amino acid sequence is shown in SEQ ID NO: 5) from the N end to The C-terminal is fused sequentially, and its amino acid sequence is shown in SEQ ID NO: 17, called "Split-AID10-N5";
- the base editing fusion protein B is composed of the second nucleobase polypeptide fragment AID10-C4 (amino acid The sequence is shown in SEQ ID NO:10), 32aa connecting peptide, the second nCas9 polypeptide fragment, 1 molecule of UGI and 1 molecule of NLS are sequentially fuse
- the system is based on the bimolecular, cytidine deaminase rA1 complementary cytosine base editing system "Split-BE3".
- the base editing fusion protein A consists of the NLS polypeptide fragment, the first nCas9 polypeptide fragment, the 32aa connecting peptide and the first nucleobase polypeptide fragment BE3-N (the amino acid sequence is shown in SEQ ID NO: 6) from the N end to The C-terminal is fused sequentially, and its amino acid sequence is shown in SEQ ID NO: 19, called "Split-BE3-N";
- the base editing fusion protein B is composed of the second nucleobase polypeptide fragment BE3-C (amino acid The sequence is shown in SEQ ID NO:11), the 32aa connecting peptide, the second nCas9 polypeptide fragment, 1 molecule of UGI and 1 molecule of NLS are sequentially fused from the N-terminus to the C-
- the system is based on the bimolecular, cytidine deaminase hA3A complementary cytosine base editing system "Split-A3A".
- the base editing fusion protein A is composed of an NLS polypeptide fragment, a first nCas9 polypeptide fragment, a 32aa connecting peptide, and a first nucleobase polypeptide fragment A3A-N (the amino acid sequence is shown in SEQ ID NO: 7).
- the base editing fusion protein B consists of the second nucleobase polypeptide fragment A3A-C (the amino acid sequence is as SEQ ID NO: Shown in 12), 32aa connecting peptide, the second nCas9 polypeptide fragment, 1 molecule of UGI and 1 molecule of NLS are sequentially fused from the N-terminus to the C-terminus. Its amino acid sequence is shown in SEQ ID NO:22, which is called "Split- A3A-C”.
- the system is based on the bimolecular, cytidine deaminase hA3B complementary cytosine base editing system "Split-A3B".
- the base editing fusion protein A consists of an NLS polypeptide fragment, a first nCas9 polypeptide fragment, a 32aa connecting peptide, and a first nucleobase polypeptide fragment A3B-N (the amino acid sequence is shown in SEQ ID NO: 8).
- the base editing fusion protein B is composed of the second nucleobase polypeptide fragment A3B-C ( The amino acid sequence is as shown in SEQ ID NO:13), the 32aa connecting peptide, the second nCas9 polypeptide fragment, 1 molecule of UGI and 1 molecule of NLS are sequentially fused from the N-terminus to the C-terminus. Its amino acid sequence is as shown in SEQ ID NO:24 Display, called "Split-A3B-C".
- the base editing system is an adenine base editor system; the nucleobase deaminase is adenosine deaminase; using this system, the system can be used in the wizard Under the guidance of RNA, one or more A bases in the target sequence are replaced with G bases.
- the adenosine deaminase is a DNA-dependent adenosine deaminase; preferably, the adenosine deaminase is a single-stranded DNA-dependent adenosine deaminase. Aminase.
- the adenosine deaminase includes a variant of E. coli tRNA adenosine deaminase TadA; preferably, the variant includes TadA-7.10, TadA-8s Or TadA-8e; more preferably, the variant is TadA-8e (International Patent: PCT/US2021/016827).
- the first nucleobase deaminase polypeptide fragment of the adenine base editing fusion protein A includes any amino acid sequence shown in SEQ ID NO: 25-26 ;
- the second nucleobase deaminase polypeptide fragment of the adenine base editing fusion protein B includes any amino acid sequence shown in SEQ ID NO: 27-28.
- the adenine base editing system includes a combination of base editing fusion protein A and base editing fusion protein B according to any one of the following 1) to 2):
- Base editing fusion protein A with the amino acid sequence shown in SEQ ID NO:29: Split-ABE8e-N
- base editing fusion protein B with the amino acid sequence shown in SEQ ID NO:30: Split-ABE8e-C ;
- Base editing fusion protein A with the amino acid sequence shown in SEQ ID NO:31: Split-ABE8e-N7
- base editing fusion protein B with the amino acid sequence shown in SEQ ID NO:32: Split-ABE8e-C2 .
- the system is based on the bimolecular, adenosine deaminase TadA-8e complementary cytosine base editing system "Split-ABE8e".
- the base editing fusion protein A consists of an NLS polypeptide fragment, a first nCas9 polypeptide fragment, a 32aa connecting peptide and a first nucleobase polypeptide fragment ABE8e-N (the amino acid sequence is shown in SEQ ID NO: 25) from the N end to The C-terminal is fused sequentially, and its amino acid sequence is shown in SEQ ID NO: 29, called "Split-ABE8e-N";
- the base editing fusion protein B is composed of the second nucleobase polypeptide fragment ABE8e-C (amino acid The sequence is shown in SEQ ID NO:27), the 32aa connecting peptide, the second nCas9 polypeptide fragment and 2 molecules of NLS are sequentially fused from the N-termin
- the system is based on the bimolecular, adenosine deaminase TadA-8e complementary cytosine base editing system "Split-ABE8e-N7-C2".
- the base editing fusion protein A consists of an NLS polypeptide fragment, a first nCas9 polypeptide fragment, a 32aa connecting peptide and a first nucleobase polypeptide fragment ABE8e-N7 (the amino acid sequence is shown in SEQ ID NO: 26) from the N end to The C-terminal is fused sequentially, and its amino acid sequence is shown in SEQ ID NO: 31, called "Split-ABE8e-N7";
- the base editing fusion protein B is composed of the second nucleobase polypeptide fragment ABE8e-C2 (amino acid The sequence is shown in SEQ ID NO:28), the 32aa connecting peptide, the second nCas9 polypeptide fragment and 2 molecules of NLS are
- the nucleotide sequence encoding base editing fusion protein A and base editing fusion protein B and/or the nucleotide sequence encoding guide RNA is consistent with the expression
- the control element is operably connected.
- the regulatory element is a promoter; the promoter includes viral 35S promoter, corn Ubi-1 promoter, rice Ubi promoter, viral CMV promoter, yeast TDH3 promoter, yeast GAL1 promoter, Arabidopsis egg cell-specific EC1.2en+EC1.1 chimeric promoter, rice U6 promoter, Arabidopsis U6 promoter or human U6 promoter.
- promoters examples include, but are not limited to, polymerase (pol) I, pol II or pol III promoters.
- the pol I promoter includes the chicken RNA pol I promoter.
- Pol II promoters include, but are not limited to, Rous sarcoma virus long terminal repeat (RSV-LTR) promoter and simian virus 40 (SV40) immediate early promoter.
- Pol III promoters include U6 and H1 promoters. Inducible promoters such as metallothionein promoters can be used.
- the promoter When used in plants, the promoter may be cauliflower mosaic virus 35S promoter, corn Ubi-1 promoter, wheat U6 promoter, rice U3 promoter, corn U3 promoter, rice actin promoter, Arabidopsis thaliana Egg cell-specific EC1.2en+EC1.1 chimeric promoter, Arabidopsis U6 promoter; when used in yeast, the promoter can be yeast ADH1 promoter, yeast TDH3 promoter, yeast GAL1 promoter, yeast SNR52 promoter promoter; when used in mammals such as humans, the promoter can be viral CMV promoter, human U3/U6 promoter.
- the present invention also provides the application of the base editing system in producing genetically modified organisms.
- the base editing system is introduced into biological cells, and the base editor is targeted to the cells by the guide RNA.
- the target sequence in the genome results in at least one C base being replaced by a T base or at least one A base being replaced by a G base in the target sequence.
- the organism is a eukaryotic organism; the eukaryotic organism is a fungus, animal or plant.
- the fungi include yeast; the animals include humans, mice, rats, monkeys, dogs, pigs, sheep, cattle or cats; the plants include monocots and gemini Leafy plants, including but not limited to Arabidopsis thaliana, rice, wheat, corn, soybean, sunflower, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava or potato.
- the method of the invention is particularly suitable for producing genetically modified plants, such as crop plants.
- the base editing system can be introduced into the plant by various methods well known to those skilled in the art. Methods that can be used to introduce the base editing system of the present invention into plants include, but are not limited to: biolistic method, PEG-mediated protoplast transformation, soil Agrobacterium-mediated transformation, plant virus-mediated transformation, pollen tube channel method, and Intraventricular injection method.
- genetically modified plants produced using a base editing system based on bimolecular deaminase complementation have excellent agronomic traits.
- the base editing system "Split-AID10" based on bimolecular deaminase complementation was used, combined with the guide RNA targeting the endogenous gene ALS of Arabidopsis thaliana, to make the endogenous target site of transgenic Arabidopsis thaliana
- a single base substitution of G 13 >A 13 occurs on the gene, resulting in an amino acid substitution from alanine to threonine (A122T) at position 122 in the amino acid sequence of the encoding gene.
- the cytosine base editing system based on bimolecular deaminase complementation of the present invention reduces the Cas9-dependent and Cas9-independent base editing on the genome. type (random type) off-target, while still maintaining relatively efficient base editing on-target activity.
- the adenine base editing system based on bimolecular deaminase complementation also shows universal applicability in almost all representative eukaryotes, in monocotyledonous plants such as rice, and dicotyledonous plants such as Pseudomonas aeruginosa.
- Arabidopsis, fungi such as yeast, and mammalian cell lines such as human HEK293T all exhibit good adenine base editing activity.
- Figure 1 is a working principle diagram of the base editing system based on bimolecular deaminase complementation.
- Figure 2 is a schematic diagram of a vector used for transient expression of base editors in rice protoplasts.
- FIG. 5 A is the base editing statistical table of three endogenous targets of Arabidopsis thaliana by the split-AID10 system; B is the use of the split-AID10 system to edit the endogenous ALS gene of Arabidopsis thaliana, thereby giving transgenic T1 generation Arabidopsis thaliana Resistance effect map and target site Sanger sequencing map of imidazolinone herbicides.
- the values of the histogram in the figure represent multiple organisms. The average of the replicates (ns, not statistically significant; ****, p value less than 0.0001).
- the base editing system based on bimolecular deaminase complementation mainly consists of three parts: base editing fusion protein A, base editing fusion protein B and guide RNA.
- the base editing fusion protein A and the base editing fusion protein B together constitute a bimolecular deaminase complementary base editor.
- the fusion protein A is formed by fusing the first nCas9 polypeptide fragment and the first deaminase polypeptide fragment through a flexible connecting peptide.
- the fusion protein B is formed by fusing the second deaminase polypeptide fragment and the second nCas9 polypeptide fragment through a flexible connecting peptide.
- the first and second nCas9 polypeptide fragments can be seamlessly fused into the complete nCas9 protein.
- the deaminase of the first and second deaminase polypeptide fragments is selected from the same nucleobase deaminase, and neither part thereof contains a complete deaminase sequence nor has deaminase activity.
- the first and second deaminase polypeptide fragments can also be Seamlessly fused into the complete corresponding nucleobase deaminase.
- the first The two deaminase polypeptide fragments have some overlapping amino acid sequences, and the length of the fused deaminase amino acid sequence exceeds that of the original selected nucleobase deaminase.
- RNPs protein nucleic acid complexes
- the examples provided by the present invention prove that the aforementioned preferred embodiments such as the Split-AID10 cytosine base editing system exhibit close to background levels of whole genome DNA in plants such as rice, fungi such as yeast, and animals such as the human HEK293T cell line.
- the off-target rate is significantly lower than that of traditional base editors such as BE3 (also called “N-BE3" in the present invention), while maintaining a relatively high DNA target efficiency.
- BE3 also called “N-BE3" in the present invention
- the aforementioned preferred embodiment Split-BE3 cytosine base editing system also showed near background levels of whole-genome DNA off-targeting in representative eukaryotic yeast and human HEK293T cell lines, which was significantly lower than that of the traditional base editor BE3. At the same time, a relatively high DNA target efficiency is maintained.
- the aforementioned preferred Split-ABE8e adenine base editing system also demonstrates a wide range of species adaptability, ranging from single-cell eukaryotic yeast, to higher plants Arabidopsis thaliana and rice, and even the human HEK293T cell line. Efficient adenine base editing of endogenous targets in eukaryotes.
- Example 1 Evaluation of on-target and off-target efficiencies of a cytosine base editing system based on bimolecular deaminase complementation in the monocotyledonous model plant rice.
- the wild-type rice variety used in this example is Oryza sativa L.ssp.japonica ZH11.
- the expression vector pHBT-rAPOBEC1-nCas9-UGI of the base editor "N-BE3" (or "BE3") was independently constructed by our laboratory.
- the promoter used is the maize ZmUbi-1 promoter and the terminator is NOS. terminator.
- the cytosine base editor "Split-AID10” based on bimolecular deaminase complementation is composed of the "Split-AID10-N” expression vector and the “Split-AID10-C” expression vector; developed by Shanghai Sangon Biotechnology Co., Ltd.
- the expression vector pHBT-Split-AID10-N was obtained from the aforementioned pHBT vector; the construction of the "Split-AID10-C" expression vector: the construction process of the pHBT-Split-AID10-C vector is basically the same as that of pHBT-Split-AID10-N, which contains the coding
- the amino acid sequence is the polynucleotide sequence of the polypeptide fragment described in SEQ ID NO: 16 (Split-AID10-C).
- the cytosine base editor "Split-AID10-N5-C4" based on bimolecular deaminase complementation is composed of the expression vector pHBT-Split-AID10-N5 and the expression vector pHBT-Split-AID10-C4.
- the construction method is the same as " Split-AID10", where the amino acid sequence of Split-AID10-N5 is as SEQ ID NO:17, and the amino acid sequence of Split-AID10-C4 is as SEQ ID NO:18.
- nSaCas9 expression vector The polynucleotide sequence of plant codon-optimized SaCas9 was a gift from Professor Puchta Holger of Germany (published paper [8] ). Using the site-directed mutagenesis kit (Mut Express II Fast Mutagenesis KitV2, #C214-01) purchased from Nanjing Vazyme Biotech Co., Ltd., the 10th position of Aspartame in the aforementioned SaCas9 encoding amino acid sequence was The amino acid was mutated into alanine to obtain the polynucleotide sequence encoding nSaCas9 (D10A). Finally, the expression vector pHBT-nSaCas9 was constructed through simple enzyme digestion and ligation method.
- MMg 0.4M mannitol, 15mM MgCl 2 and 4mM MES pH5.7
- 8 endogenous rice target sites were selected, including 4 on-target sites targeted by the SpCas9 guide RNA (OsAAT1, OsCDC48, OsNAL1 and OsPDS1), and 4 off-target sites targeted by the nSaCas9 guide RNA. points (OsCDC48, OsNRT1.1B, OsDEP1, OsAAT1).
- the aforementioned target sites were randomly grouped according to the one-to-one correspondence between SpCas9 and SaCas9, and Sp-OsAAT1 and Sa-OsCDC48, Sp-OsCDC48 and Sa-OsNRT1.1B were obtained.
- Sp-OsNAL1 and Sa-OsDEP1 and Sp-OsPDS1 and Sa-OsAAT1 were randomly grouped according to the one-to-one correspondence between SpCas9 and SaCas9, and Sp-OsAAT1 and Sa-OsCDC48, Sp-OsCDC48 and Sa-OsNRT1.1B.
- the genetic transformation vector of base editor BE3 is pH-nCas9-PBE (addgene: #98163), in which the promoter used is the ZmUbi-1 promoter and the terminator used is the E9 terminator.
- Targeted editing of the target gene OsSWEET11/13/14 The expression cassettes of three guide RNAs, OsU6apro-OsSWEET14-OsU6bpro-OsSWEET13-OsU6apro-OsSWEET11, were commercially synthesized and fused into the aforementioned pH-nCas9-PBE vector (target Sequence information is listed in Table 1).
- Targeted editing of the target gene OsSLR1 a commercially synthesized expression cassette of a single guide RNA, OsU6apro-OsSLR1, was fused into the aforementioned pH-nCas9-PBE vector through simple enzyme digestion (target sequence information is listed in Table 1);
- the average C to T base substitution efficiency is about 4.6% and 6.6%, which is greater than 3.3% of the traditional base editor N-BE3 (BE3) ( Figure 3A, 3C).
- off-target editing deep sequencing analysis was performed on 4 nSaCas9 guide RNA targets. The results showed that the average off-target editing efficiencies (0.05% and 0.8%) of Split-AID10 and Split-AID10-N5C4 at the 4 sites were significantly lower than Traditional base editor BE3 (1.6%).
- its average off-target efficiency is only 0.05%, which is basically consistent with the 0.02% of the blank control ( Figure 3B, 3C).
- the Split-AID10 base editor with the best performance in the aforementioned protoplasts was fused with the guide RNA to construct a genetic transformation vector for genetic transformation of rice.
- the base editor was evaluated in rice transgenic plants, and traditional base editing was selected.
- Device N-BE3 was used as a control.
- specific primers were designed for different target gene sites for PCR, and the products were sent to Tsingke Biotech.Co., Ltd. for Sanger sequencing.
- the analysis results for four targets showed that the base editor Split-AID10 also successfully performed base editing in transgenic plants.
- the effective editing window was from C 5 to C 13 of the target sequence (using PAM as 21-23 ), the cytosine base substitution efficiency at different positions ranged from 3% to 55.2% ( Figure 3A).
- CRISPR-GE http://skl.scau.edu.cn/
- Sanger sequencing of the two off-target sites was performed on a total of 38 transgenic rice plants that were successfully edited at this site using three base editors, BE3 and Split-AID10. As shown in Figure 3B, Split-AID10 only found the aforementioned Cas9-dependent DNA off-target situation in an average of 50% of edited plants, which was significantly less than 100% of BE3.
- split-AID10 two cytosine base editing systems based on bimolecular deaminase complementation
- BE3 traditional base editors
- Split-AID10 also demonstrated good base editing on-target efficiency at the level of transgenic rice plants, and was also found to reduce the probability of Cas9-dependent off-target occurrence.
- Example 2 Using the cytosine base editing system Split-AID10 based on bimolecular deaminase complementation can also achieve efficient base editing in transgenic Arabidopsis plants.
- the wild-type Arabidopsis used in this example is Col-0 ecotype (Arabidopsis thaliana Col-0).
- the Arabidopsis thaliana genetic transformation vector used in this example was constructed with reference to the published article [15] and the method described in Example 1. Genetic transformation vectors were constructed for two different targets (see Table 1 for guide RNA target sequence information). The aforementioned vectors were transformed into Agrobacterium tumefaciens strain GV3101 by electroporation, and Arabidopsis plants were transformed using the pollen tube introduction method. Specifically, the GV3101 bacterial solution containing the target vector was inoculated into liquid LB medium containing kanamycin (50 mg/L) at a ratio of 1:100, and cultured in a 28°C shaker at a rotation speed of 220 rpm for 2 days.
- Collect the bacterial cells at 5000g discard the medium, add 5% sucrose solution containing 0.05% Silwet L77 and resuspend. Take the flowering Arabidopsis plant, turn it upside down and completely immerse the inflorescence in the Agrobacterium bacteria solution. Stir gently for about 10 seconds, take it out, place it in a moist dark environment for 1 day, and then transfer it to a normal growth environment until mature seeds are harvested.
- the mature seeds obtained above were sown in a sterile 1/2MS solid medium containing 50 mg/L hygromycin B in a sterile environment. About 10 days later, the rooted hygromycin-positive seedlings were moved to soil to resume culture. Half a month later, the leaf genome of the positive seedlings was extracted using a fast plant genomic DNA extraction system purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Target gene-specific primers were designed for PCR and sent to Qingke Company for Sanger sequencing.
- Example 3 The cytosine base editing systems Split-AID10 and Split-BE3 based on bimolecular deaminase complementation also exhibit good on-target editing and extremely low genome-wide random off-targeting in single-cell eukaryotic yeast.
- Saccharomyces cerevisiae BY4741 strain was used in this example.
- the yeast U6 promoter is directly PCR amplified from the genome, and then the complete expression original of the guide RNA is assembled through an overlapping PCR strategy to complete a series of guide RNA vectors to construct pGAL1-yGFP-SNR35p-sgRNAs.
- table 3 For information on guide RNA target points in yeast, see table 3.
- Target sequence PolyC-1 CCCCCCATGTTCCGAGAT CGG PolyC-2 TCCCCCCCCTCAATTCCAGC AGG PolyC-3 ATCAGCCCCCCCCCCAAGGAA AGG PolyC-4 GAACAGCTGAACCCCCCCAA TGG PolyC-5 CATTAAAGCAACCCCCCATA GGG Can1-5 TCCAATAACGGAATCCAACT GGG
- Saccharomyces cerevisiae used was based on published articles [17] .
- the aforementioned vector was transferred into Saccharomyces cerevisiae BY4741 strain through conventional lithium acetate LiOAC chemical transformation method, and the auxotrophic genes on the plasmid were used to screen positive transformants.
- the positive transformed clones containing the target plasmid were picked from the auxotrophic plate into liquid deficiency medium, cultured in a medium with 2% glucose carbon source for two days until saturated, and then inoculated into 2 cells at a dilution ratio of 1:1000.
- % raffinose carbon source medium was cultured for two days until saturation, and finally inoculated into 1% galactose carbon source medium at a dilution ratio of 1:10,000 and cultured for two to three days until saturation, and an appropriate amount of bacterial fluid was collected for subsequent genome extraction.
- the analysis results of 6 endogenous targets in the yeast genome show that the average editing efficiency of the base editor N-BE3 is 6.5% to 94.9%, and the effective editing window is from C 3 to C 10 of the target sequence. position; the average editing efficiency of the base editor Split-AID10 is 56.9% to 97.3%, and the effective editing window is C 5 to C 15 of the target sequence; the average editing efficiency of the base editor Split-BE3 is 57.8% to 96.8% , the effective editing window is C 5 to C 14 of the target sequence.
- the editing windows of Split-AID10 and Split-BE3 are both shifted and closer to the PAM recognition region.
- bimolecular deaminase complementary base editing system has efficient editing activity in the eukaryotic cell Saccharomyces cerevisiae. At the same time, it can significantly reduce genome-wide off-target effects and has very high safety performance.
- Example 4 The Split-AID10 and Split-BE3 systems based on bimolecular cytidine deaminase complementation also demonstrated good on-target editing and extremely low genome-wide random off-targeting in the human embryonic kidney cell line 293T.
- Human embryonic kidney cell line 293T (HEK293T) was obtained from the American Type Culture Collection (ATCC).
- the core vector integrates the CMV promoter and the human U6 promoter.
- the vector backbone is pX330 (Addgene#42230), and then the relevant base editor pCMV-rAPOBEC1-nCas9-UGI(N -BE3), pCMV-Split-AID10 and pCMV-Split-BE3 expression vectors, where the Split-AID10-N and Split-AID10-C amino acid sequences contained in the pCMV-Split-AID10 expression vector are as described above, pCMV-Split -The Split-BE3-N and Split-BE3-C amino acid sequences contained in the BE3 expression vector are also as described above; in this example, the guide RNA expression vector is modified as follows, and the red fluorescent protein mScarlet is inserted downstream of CMV for indication.
- HEK293T cells are human embryonic kidney cells that grow adherently and are cultured in DMEM high-glucose medium and 10% fetal calf serum in an incubator at 37°C and 5% carbon dioxide.
- DMEM high-glucose medium fetal calf serum
- a suitable number of cells was spread in a 48-well plate.
- conventional liposomes were used for cell transfection. After 48-72 hours of expression, the cells were collected and genome extracted.
- R-Loop detection uses pX601 (Addgene#61591) as an expression vector.
- the expression vector pX601 has been appropriately modified, including the use of nSaCas9 nickase and the addition of a uracil glycosylase inhibitory functional domain.
- the target site information of UGI and orthogonal detection is detailed in Table 4.
- Deep sequencing library construction adopts a two-step strategy: 1) First, design target-specific binding primers for capture, and carry a partial sequence of the Illumina Nextera adapter at the end of the primer; 2) Then use the PCR product of the first reaction as a template , perform amplification of the complete library, including the 8-base sample barcode and the complete sequence of P5/P7. Finally, the products of the two rounds of PCR amplification were subjected to a series of routine operations such as purification, concentration determination, and library homogenization and mixing, and were sent to Suzhou Genewiz Inc. (sz) for high-throughput sequencing.
- routine operations such as purification, concentration determination, and library homogenization and mixing
- the analysis of amplicon deep sequencing results mainly includes two parts: library splitting and editing efficiency calculation.
- Library splitting is carried out using Illumina's official bcl2fastq software.
- the original BCL data that are downloaded are split into independent FASTQ format files one by one according to the barcode table of the mixed sample, including read length 1 (Read1) and read length 2 (Read2); then CRISPResso2 [12] analysis is performed separately for each target point to obtain a matrix of base substitutions near the target point, and finally the editing efficiency is calculated.
- This example also performs Cas9 protein-dependent off-target detection for 4 targets.
- the prediction software Cas-OFFinder is used to perform a fault-tolerant search of less than 6 for each target sequence, and searches for Cas9 protein-dependent off-targets from the entire human genome. position; then screen based on the core seed region of the CRIPSR/Cas9 system to retain potential off-target site sequences that are 5 bases in length close to the PAM region and have no mismatches; finally, a relatively simple set of sequences is designed based on a large number of published literature and methods.
- the scoring algorithm analyzes the sequence characteristics of potential off-target sites, further selects sites with higher scores, and designs specific capture primers for subsequent library construction and sequencing of amplicons.
- bimolecular deaminase complementary base editing system has efficient editing activity in human HEK293T cells. At the same time, it can not only reduce Cas9-dependent off-target editing, but also significantly reduce genome-wide off-target effects. , showing excellent safety performance and having broad practical application prospects.
- the expression vectors of the Split-A3A system are pGAL1-Split-A3A-N and pGAL1-Split-A3A-C; the amino acid sequence encoding Split-A3A-N is shown in SEQ ID NO: 10, The amino acid sequence encoding Split-A3A-C is shown in SEQ ID NO: 11; the expression vectors of the Split-A3B system are pGAL1-Split-A3B-N and pGAL1-Split-A3B-C, encoding the amino acid of Split-A3B-N The sequence is shown in SEQ ID NO: 12, and the amino acid sequence encoding Split-A3B-C is shown in SEQ ID NO: 13.
- the Split-A3A system based on cytidine deaminase A3A and the Split-A3B system based on cytidine deaminase A3B successfully realized the base of the target sequence C to T in representative eukaryotic yeast. Base replacement.
- the editing window of the Split-A3A system is from C 1 to C 15 , and the editing efficiency of C bases at different positions in the editing window is 22.1% to 84.7%; the editing window of the Split-A3B system is from C 5 to C 15 in the editing window.
- the editing efficiency of C bases at different positions within the gene ranges from 9.5% to 95.5%.
- Example 6 The adenine base editing systems Split-ABE8e and Split-ABE8e-N7-C2 based on bimolecular deaminase complementation can achieve efficient adenine base editing in plants.
- the rice material is the same as Example 1, and the Arabidopsis material is the same as Example 2.
- the Arabidopsis transient expression vectors of traditional adenine base editors ABE7.10 and ABE8e are pHBT-ABE7.10 and pHBT-ABE8e, in which the promoter used is AtUBQ10 promoter and the terminator is NOS terminator.
- the aforementioned vectors were all constructed by our laboratory and have been published [15] ; the expression vectors of the Split-ABE8e system and Split-ABE8e-N7-C2 system were constructed with reference to the Split-AID10 system and Split-AID10-N5- described in Example 1.
- the construction method of the C4 system is to obtain the pHBT-Split-ABE8e-N vector and pHBT-Split-ABE8e-C vector of the Split-ABE8e system and the pHBT of the Split-ABE8e-N7-C2 system based on the pHBT-PIGS-ABE8e vector. -Split-ABE8e-N7 vector and pHBT-Split-ABE8e-C2 vector.
- the amino acid sequence encoding Split-ABE8e-N is such as SEQ NO:29
- the amino acid sequence encoding Split-ABE8e-C is such as SEQ NO:30
- the amino acid sequence encoding Split-ABE8e-N7 is such as SEQ NO:31
- the amino acid sequence of ABE8e-C2 is as SEQ NO: 32
- the guide RNA transient expression vector suitable for Arabidopsis is based on Li, Z. et al. 2019. Current protocols in molecular biology, https://doi.org/10.1002/cpmb. 89 published the article pUC119-AtU6-26pro-sgRNA) vector to construct the pUC119-AtU6-26pro-AtFLS2-sgRNA expression vector (see Table 1 for the target sequence).
- Example 1 The rice genetic transformation method is described in Example 1, and the Arabidopsis thaliana genetic transformation method is described in Example 2.
- the data provided in this example prove that the adenine base editing systems Split-ABE8e and Split-ABE8e-N7-C2 based on bimolecular deaminase complementation can effectively perform adenine base editing in plants.
- the Split-ABE8e system can perform efficient adenine base editing of endogenous target genes in transgenic plants of the representative monocotyledonous plant rice and the representative dicotyledonous plant Arabidopsis thaliana, revealing the widespread use of this system in plants. applicability.
- Example 7 The Split-ABE8e system can achieve efficient adenine base editing in yeast and human embryonic kidney cell line HEK293T.
- Example 3 For the experimental materials and methods implemented in yeast, refer to Example 3; for the experimental materials and methods implemented in the HEK293T cell line, refer to Example 4.
- the expression vectors of the Split-ABE8e system in yeast are pGAL1-Split-ABE8e-N and pGAL1-Split-ABE8e-C, and the other control vectors are pGAL1-nSpCas9, pGAL1-N-ABE7.10 and pGAL1-N-ABE8e.
- the expression vectors of the Split-ABE8e system in the HEK293T cell line are pCMV-Split-ABE8e-N and pCMV-Split-ABE8e-C; the aforementioned yeast and human Split-ABE8e expression vectors contain Split-ABE8e-N and Split-
- the encoded amino acid sequence of ABE8e-C is as described above; the guide RNA expression vectors in yeast and human cells are as described in Examples 3 and 4, and the guide RNA target sequence information is shown in Tables 3 and 4.
- FIG. 10A shows the results of the Split-ABE8e system and the guide RNA targeting the yeast endogenous Can1-8 site.
- the results are shown in Figure 10A.
- the Split-ABE8e system was successfully used in yeast.
- Adenine base editing was performed on the Can1-8 site, and the editing window was A 4 -A 13 .
- the substitution efficiency from A base to G base ranged from 25% to 82%.
- Figure 10B shows the successful editing of another endogenous site Can1-9 in yeast cells by the Split-ABE8e system.
- the Split-ABE8e system and the guide RNA targeting the endogenous HEK Site 1 site were then co-transfected into the HEK293T cell line.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Enzymes And Modification Thereof (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
一种基于双分子脱氨酶互补的碱基编辑系统及其应用，主要由nCas9和核碱基脱氨酶双重互补的碱基编辑融合蛋白A、B以及向导RNA组成，在保持高效胞嘧啶碱基编辑中靶效率的同时，极大降低了其在基因组上的Cas9依赖型和Cas9非依赖型脱靶。还提供了基于双分子脱氨酶互补的腺嘌呤碱基编辑系统。基于双分子脱氨酶互补的胞嘧啶和腺嘌呤碱基编辑系统普遍适用于各种真核生物，可用于作物遗传育种、动物品种改良乃至人类遗传疾病的临床治疗等领域。
Description
本发明涉及基于双分子脱氨酶互补的碱基编辑系统及其应用，属于基因工程技术领域。
数量众多的作物农艺性状以及人类遗传疾病由基因组上的单核苷酸多态性(Single nucleotide polymorphisms,SNPs)决定
[1,2]。由CRISPR/Cas9基因编辑技术衍生而来的碱基编辑技术(Base editing)具有不产生DNA双链断裂以及可精确实现靶位点单碱基替换等特点，极大提高了基因编辑的精确度和安全性，因而被广泛应用于基因功能研究、作物遗传改良和人类基因疾病的临床治疗等领域
[3]。碱基编辑系统主要由碱基编辑器(Base editors,BEs)和向导RNA(single-guide RNA,sgRNA)构成。科研人员最常用的碱基编辑器BE3是将来自大鼠的胞苷脱氨酶rAPOBEC1、来自噬菌体的尿嘧啶糖苷酶抑制子(Uracil glycosylase inhibitor,UGI)与Cas9切口酶(Cas9 nickase,nCas9-D10A)融合获得
[4]。在sgRNA的引导下，BE3结合于靶标基因位点处，其所融合的胞苷脱氨酶rAPOBEC1发挥脱氨作用，将靶序列中特定位置的胞嘧啶(C)碱基突变为尿嘧啶(U)碱基。在细胞内源修复机制的作用下，U碱基变为胸腺嘧啶(T)碱基，最终达到C到T的碱基替换
[4]。
然而，近年来国际上多个在水稻、小鼠等生物体中的独立研究均发现，传统碱基编辑系统(如BE3系统)会在全基因组水平上引发大量不依赖于向导RNA(或Cas9)的随机脱靶突变
[5,6]。这给碱基编辑系统的实际使用，增加了难以评估的安全隐患，尤其是给临床应用带来了极大的不确定性。因此，发展高效、通用且安全的低脱靶碱基编辑系统，对基础科学研究、作物遗传改良乃至人类基因疾病的临床治疗都将大有裨益。
发明内容
本发明的目的在于提供一种新型的基于双分子脱氨酶互补的碱基编辑系统及其应用，本发明提出的基于双分子脱氨酶互补的碱基编辑系统能极大减少传统碱基编辑系统存在的全基因组随机脱靶问题，同时仍保持了高效的中靶编辑，具有广阔的应用前景。
为实现上述目的，本发明采取的技术方案为：一种双分子脱氨酶互补的碱基编辑系统，所述碱基编辑系统包括以下1)至5)中至少一项：
1)碱基编辑融合蛋白A、碱基编辑融合蛋白B和向导RNA；
2)包含编码碱基编辑融合蛋白A和碱基编辑融合蛋白B的核苷酸序列的表达构建体，和向导RNA；
3)碱基编辑融合蛋白A、碱基编辑融合蛋白B、和包含编码向导RNA的核苷酸序列的表达构建体；
4)包含编码碱基编辑融合蛋白A和碱基编辑融合蛋白B的核苷酸序列的表达构建体，和包含编码向导RNA的核苷酸序列的表达构建体；
5)包含编码碱基编辑融合蛋白A和碱基编辑融合蛋白B的核苷酸序列和编码向导RNA的核苷酸序列的表达构建体；
其中所述的碱基编辑融合蛋白A自N端到C端依次包括第一nCas9多肽片段、柔性连接肽和第一核碱基脱氨酶多肽片段；所述碱基编辑融合蛋白B自N端到C端依次包括第二核碱基脱氨酶多肽片段、柔性连接肽和第二nCas9多肽片段；所述第一核碱基脱氨酶多肽片段和第二核碱基脱氨酶多肽片段选自相同的核碱基脱氨酶。
需要指出的是，“表达构建体”指适于感兴趣的核苷酸序列在生物体中表达的载体如重组载体。“表达”指可产生有功能的产物。例如，核苷酸序列的表达可指核苷酸序列的转录(如转录生成mRNA或功能RNA)和/或RNA翻译成前体或成熟蛋白质。本发明所述“表达构建体”可以是线性的核酸片段、环状质粒、病毒载体，或者可以是能够翻译的RNA(如mRNA)。本发明的“表达构建体”可包含相同来源但以不同于天然排列方式的调控序列和感兴趣的核苷酸序列，亦包含不同来源的调控序列和感兴趣的核苷酸序列。
本申请发明人发现，在本发明所述的碱基编辑系统中，向导RNA能与碱基编辑融合蛋白A和碱基编辑融合蛋白B形成蛋白核酸复合体(RNP)，并引导RNP靶向目标DNA序列，导致所述靶标序列中一至多个胞嘧啶碱基到胸腺嘧啶碱基，或一至多个腺嘌呤(A)碱基到鸟嘌呤(G)碱基的核苷酸取代。且其第一核碱基脱氨酶多肽片段和第二核碱基脱氨酶多肽片段均不含有完整脱氨酶序列，也不具有碱基脱氨活性，这是该系统降低脱靶的关键所在。
本申请发明人发现，在本发明所述的碱基编辑系统中，所述第一、第二核碱基脱氨酶通常是互补的，即可融合成具备完整脱氨活性的核碱基脱氨酶。
作为本发明所述碱基编辑系统的优选实施方式，所述第一nCas9多肽片段的氨基酸序列如SEQ ID NO:1所示；所述第二nCas9多肽片段的氨基酸序列如SEQ ID NO:2所示。
作为本发明所述碱基编辑系统的优选实施方式，所述柔性连接肽包括氨基酸序列如SEQ ID NO:3所示的32aa连接肽。研究表明，所述柔性连接肽还包括XTEN、PR、GGGGS、PRGGSGG、ARGGSGG、GS、GAP、(GGGGS)×3、GGS和(GGS)×7。所述柔性的连接肽也可以是长1-50个或更多个氨基酸、无二级以上结构的非功能性氨基酸序列。
作为本发明所述碱基编辑系统的优选实施方式，所述碱基编辑融合蛋白A和碱基编辑融合蛋白B的N端或C端融合有至少一个核定位序列。研究表明，所述核定位序列(NLS)通常可以与入核载体相互作用，从而可以使靶蛋白能够被运送进细胞核。一般而言，NLS由暴露于蛋白表面上的带正电的赖氨酸或精氨酸的一个或多个短序列组成，但其他类型的NLS也是已知的。NLS的非限制性实例包括氨基酸序列如：PKKKRKV或KRPAATKKAGQAKKKK。在一些具体的实施例中，本发明所述碱基编辑融合蛋白A的N端包含1个拷贝的NLS(氨基酸序列为PKKKRKV),所述碱基编辑融合蛋白B的C端包含1个拷贝的NLS(氨基酸序列为KRPAATKKAGQAKKKK)或2个拷贝的NLS(氨基酸序列为PKKKRKV和KRPAATKKAGQAKKKK)。
作为本发明所述碱基编辑系统的优选实施方式，所述碱基编辑系统为胞嘧 啶碱基编辑器系统；所述核碱基脱氨酶为胞苷脱氨酶；利用该系统可在向导RNA的引导下使目标序列中的一至多个C碱基替换为T碱基。
作为本发明所述碱基编辑系统的优选实施方式，所述胞苷脱氨酶选自：由APOBEC3A(A3A)、APOBEC3B(A3B)、APOBEC3C(A3C)、APOBEC3D(A3D)、APOBEC3F(A3F)、APOBEC3G(A3G)、APOBEC3H(A3H)、APOBEC1(A1)、APOBEC3(A3)、APOBEC2(A2)、APOBEC4(A4)和AICDA(AID)或通过单个或多个氨基酸组合突变APOBEC3A(A3A)、APOBEC3B(A3B)、APOBEC3C(A3C)、APOBEC3D(A3D)、APOBEC3F(A3F)、APOBEC3G(A3G)、APOBEC3H(A3H)、APOBEC1(A1)、APOBEC3(A3)、APOBEC2(A2)、APOBEC4(A4)和AICDA(AID)获得的胞苷脱氨酶变体构成的组。
作为本发明所述碱基编辑系统的优选实施方式，所述胞苷脱氨酶选自人源AID高活性变体AID10(中国专利：202010285948.1)、大鼠来源的rA1、人源的hA3A或人源的hA3B。
作为本发明所述碱基编辑系统的优选实施方式，所述胞嘧啶碱基编辑融合蛋白A的第一核碱基脱氨酶多肽片段包括如SEQ ID NO:4～8所示任一氨基酸序列；所述胞嘧啶碱基编辑融合蛋白B的第二核碱基脱氨酶多肽片段包括如SEQ ID NO:9～13所示任一氨基酸序列。
作为本发明所述碱基编辑系统的优选实施方式，所述胞嘧啶碱基编辑融合蛋白B的C端融合有至少一个尿嘧啶糖苷酶抑制子(UGI)，所述UGI的氨基酸序列如如SEQ ID NO:14所示
作为本发明所述碱基编辑系统的优选实施方式，所述胞嘧啶碱基编辑系统包括以下1)至5)任一项所述的碱基编辑融合蛋白A和碱基编辑融合蛋白B的组合：
1)氨基酸序列如SEQ ID NO:15所示的碱基编辑融合蛋白A：Split-AID10-N，和氨基酸序列如SEQ ID NO:16所示的碱基编辑融合蛋白B：Split-AID10-C；
2)氨基酸序列如SEQ ID NO:17所示的碱基编辑融合蛋白A：Split-AID10-N5，和氨基酸序列如SEQ ID NO:18所示的碱基编辑融合蛋白B： Split-AID10-C4；
3)氨基酸序列如SEQ ID NO:19所示的碱基编辑融合蛋白A：Split-BE3-N，和氨基酸序列如SEQ ID NO:20所示的碱基编辑融合蛋白B：Split-BE3-C；
4)氨基酸序列如SEQ ID NO:21所示的碱基编辑融合蛋白A：Split-A3A-N，和氨基酸序列如SEQ ID NO:22所示的碱基编辑融合蛋白B：Split-A3A-C；
5)氨基酸序列如SEQ ID NO:23所示的碱基编辑融合蛋白A：Split-A3B-N，和氨基酸序列如SEQ ID NO:24所示的碱基编辑融合蛋白B：Split-A3B-C。
在本发明提供的具体实施方式中，所述的系统是基于双分子的、胞苷脱氨酶AID10互补的胞嘧啶碱基编辑系统“Split-AID10”。其所述碱基编辑融合蛋白A由NLS多肽片段、第一nCas9多肽片段(氨基酸序列如SEQ ID NO:1所示)、32aa连接肽(氨基酸序列如SEQ ID NO:3所示)以及第一核碱基多肽片段AID10-N(氨基酸序列如SEQ ID NO:4所示)由N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:15所示，称为“Split-AID10-N”；所述碱基编辑融合蛋白B由第二核碱基多肽片段AID10-C(氨基酸序列如SEQ ID NO:9所示)、32aa连接肽、第二nCas9多肽片段(氨基酸序列如SEQ ID NO:2所示)、1分子UGI(氨基酸序列如SEQ ID NO:14所示)和1分子NLS从N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:16所示，称为“Split-AID10-C”(如图2所示)。
在本发明提供的具体实施方式中，所述的系统是基于双分子的、胞苷脱氨酶AID10互补的胞嘧啶碱基编辑系统“Split-AID10-N5-C4”。其所述碱基编辑融合蛋白A由NLS多肽片段、第一nCas9多肽片段、32aa连接肽以及第一核碱基多肽片段AID10-N5(氨基酸序列如SEQ ID NO:5所示)由N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:17所示，称为“Split-AID10-N5”；所述碱基编辑融合蛋白B由第二核碱基多肽片段AID10-C4(氨基酸序列如SEQ ID NO:10所示)、32aa连接肽、第二nCas9多肽片段、1分子UGI和1分子NLS从N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:18所示，称为“Split-AID10-C4”(如图2所示)。
在本发明提供的具体实施方式中，所述的系统是基于双分子的、胞苷脱氨酶rA1互补的胞嘧啶碱基编辑系统“Split-BE3”。其所述碱基编辑融合蛋白A 由NLS多肽片段、第一nCas9多肽片段、32aa连接肽以及第一核碱基多肽片段BE3-N(氨基酸序列如SEQ ID NO:6所示)由N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:19所示，称为“Split-BE3-N”；所述碱基编辑融合蛋白B由第二核碱基多肽片段BE3-C(氨基酸序列如SEQ ID NO:11所示)、32aa连接肽、第二nCas9多肽片段、1分子UGI和1分子NLS从N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:20所示，称为“Split-BE3-C”。
在本发明提供的具体实施方式中，所述的系统是基于双分子的、胞苷脱氨酶hA3A互补的胞嘧啶碱基编辑系统“Split-A3A”。其所述碱基编辑融合蛋白A由NLS多肽片段，第一nCas9多肽片段、32aa连接肽以及第一核碱基多肽片段A3A-N(氨基酸序列如SEQ ID NO:7所示)依次融合而成，其氨基酸序列如SEQ ID NO:21所示，称为“Split-A3A-N”；所述碱基编辑融合蛋白B由第二核碱基多肽片段A3A-C(氨基酸序列如SEQ ID NO:12所示)、32aa连接肽、第二nCas9多肽片段、1分子UGI和1分子NLS从N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:22所示，称为“Split-A3A-C”。
在本发明提供的具体实施方式中，所述的系统是基于双分子的、胞苷脱氨酶hA3B互补的胞嘧啶碱基编辑系统“Split-A3B”。其中，所述碱基编辑融合蛋白A由NLS多肽片段、第一nCas9多肽片段、32aa连接肽以及第一核碱基多肽片段A3B-N(氨基酸序列如SEQ ID NO:8所示)由N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:23所示，称为“Split-A3B-N”；所述碱基编辑融合蛋白B由第二核碱基多肽片段A3B-C(氨基酸序列如SEQ ID NO:13所示)、32aa连接肽、第二nCas9多肽片段、1分子UGI和1分子NLS从N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:24所示，称为“Split-A3B-C”。
作为本发明所述碱基编辑系统的优选实施方式，所述碱基编辑系统为腺嘌呤碱基编辑器系统；所述核碱基脱氨酶为腺苷脱氨酶；利用该系统可在向导RNA的引导下使目标序列中的一至多个A碱基替换为G碱基。
作为本发明所述碱基编辑系统的优选实施方式，所述腺苷脱氨酶为DNA依赖型腺苷脱氨酶；优选地，所述腺苷脱氨酶为单链DNA依赖型腺苷脱氨酶。
作为本发明所述碱基编辑系统的优选实施方式，所述腺苷脱氨酶包括大肠杆菌tRNA腺苷脱氨酶TadA的变体；优选地，所述变体包括TadA-7.10、TadA-8s或TadA-8e；更优选地，所述变体为TadA-8e(国际专利：PCT/US2021/016827)。
作为本发明所述碱基编辑系统的优选实施方式，所述腺嘌呤碱基编辑融合蛋白A的第一核碱基脱氨酶多肽片段包括如SEQ ID NO:25～26所示任一氨基酸序列；所述腺嘌呤碱基编辑融合蛋白B的第二核碱基脱氨酶多肽片段包括如SEQ ID NO:27～28所示任一氨基酸序列。
作为本发明所述碱基编辑系统的优选实施方式，所述腺嘌呤碱基编辑系统包括以下1)至2)任一项所述的碱基编辑融合蛋白A和碱基编辑融合蛋白B组合：
1)氨基酸序列如SEQ ID NO:29所示的碱基编辑融合蛋白A：Split-ABE8e-N，和氨基酸序列如SEQ ID NO:30所示的碱基编辑融合蛋白B：Split-ABE8e-C；
2)氨基酸序列如SEQ ID NO:31所示的碱基编辑融合蛋白A：Split-ABE8e-N7，和氨基酸序列如SEQ ID NO:32所示的碱基编辑融合蛋白B：Split-ABE8e-C2。
在本发明提供的具体实施方式中，所述的系统是基于双分子的、腺苷脱氨酶TadA-8e互补的胞嘧啶碱基编辑系统“Split-ABE8e”。其所述碱基编辑融合蛋白A由NLS多肽片段、第一nCas9多肽片段、32aa连接肽以及第一核碱基多肽片段ABE8e-N(氨基酸序列如SEQ ID NO:25所示)由N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:29所示，称为“Split-ABE8e-N”；所述碱基编辑融合蛋白B由第二核碱基多肽片段ABE8e-C(氨基酸序列如SEQ ID NO:27所示)、32aa连接肽、第二nCas9多肽片段和2分子NLS从N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:30所示，称为“Split-ABE8e-C”。
在本发明提供的具体实施方式中，所述的系统是基于双分子的、腺苷脱氨酶TadA-8e互补的胞嘧啶碱基编辑系统“Split-ABE8e-N7-C2”。其所述碱基编辑融合蛋白A由NLS多肽片段、第一nCas9多肽片段、32aa连接肽以及第一核碱基多肽片段ABE8e-N7(氨基酸序列如SEQ ID NO:26所示)由N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:31所示，称为“Split-ABE8e-N7”； 所述碱基编辑融合蛋白B由第二核碱基多肽片段ABE8e-C2(氨基酸序列如SEQ ID NO:28所示)、32aa连接肽、第二nCas9多肽片段和2分子NLS从N端到C端依次融合而成，其氨基酸序列如SEQ ID NO:32所示，称为“Split-ABE8e-C2”。
作为本发明所述碱基编辑系统的优选实施方式，所述编码碱基编辑融合蛋白A和碱基编辑融合蛋白B的核苷酸序列和/或所述编码向导RNA的核苷酸序列与表达调控元件可操作地连接。
作为本发明所述碱基编辑系统的优选实施方式，所述调控元件为启动子；所述启动子包括病毒35S启动子，玉米Ubi-1启动子，水稻Ubi启动子，病毒CMV启动子，酵母TDH3启动子，酵母GAL1启动子，拟南芥卵细胞特异的EC1.2en+EC1.1嵌合启动子，水稻U6启动子，拟南芥U6启动子或人U6启动子。
研究表明，本发明可使用的启动子的实例包括但不限于聚合酶(pol)I、pol II或pol III启动子。pol I启动子的包括鸡RNApol I启动子。pol II启动子包括但不限于劳斯肉瘤病毒长末端重复(RSV-LTR)启动子和猿猴病毒40(SV40)立即早期启动子。pol III启动子包括U6和H1启动子。可以使用诱导型启动子如金属硫蛋白启动子。当用于植物时，启动子可以是花椰菜花叶病毒35S启动子、玉米Ubi-1启动子、小麦U6启动子、水稻U3启动子、玉米U3启动子、水稻肌动蛋白启动子,拟南芥卵细胞特异的EC1.2en+EC1.1嵌合启动子、拟南芥U6启动子；当用于酵母时，启动子可以是酵母ADH1启动子，酵母TDH3启动子，酵母GAL1启动子，酵母SNR52启动子；当用于人等哺乳动物时，启动子可以是病毒CMV启动子，人U3/U6启动子。
本发明还提供所述的碱基编辑系统在产生经遗传修饰的生物体中的应用，将所述碱基编辑系统导入生物体细胞，由所述向导RNA将所述碱基编辑器靶向细胞基因组中的靶序列，导致所述靶序列中至少一个C碱基被T碱基取代或至少一个A碱基被G碱基取代。
作为本发明所述应用的优选实施方式，所述生物为真核生物；所述真核生物为真菌、动物或植物。
作为本发明所述应用的优选实施方式，所述真菌包括酵母；所述动物包括人、小鼠、大鼠、猴、犬、猪、羊、牛或猫；所述植物包含单子叶植物和双子叶植物，包括并不限于拟南芥、水稻、小麦、玉米、大豆、向日葵、高粱、油菜、苜蓿、棉花、大麦、粟、甘蔗、番茄、烟草、木薯或马铃薯。
本发明的方法尤其适合于产生经遗传修饰的植物，例如作物植物。在本发明的产生经遗传修饰的植物的方法中，所述碱基编辑系统可以本领域技术人员熟知的各种方法导入植物。可用于将本发明的碱基编辑系统导入植物的方法包括但不限于：基因枪法、PEG介导的原生质体转化、土壤农杆菌介导的转化、植物病毒介导的转化、花粉管通道法和子房注射法。
在本发明提供的一些优选地具体实施方式中，使用基于双分子脱氨酶互补的碱基编辑系统产生的经遗传修饰的植物体具有优良的农艺性状。如图5B所示，使用基于双分子脱氨酶互补的碱基编辑系统“Split-AID10”，结合靶向拟南芥内源基因ALS的向导RNA，使转基因拟南芥该内源靶位点上发生G
13>A
13的单碱基替换，从而导致该编码基因的氨基酸序列发生第122位丙氨酸到苏氨酸(A122T)的氨基酸替换。后续的除草剂喷施实验表明，含有该A122T氨基酸替换的转基因拟南芥植株均对咪唑啉酮类除草剂产生了抗性，而不含该氨基酸替换的拟南芥植株全部枯萎致死，说明使用Split-AID10碱基编辑系统赋予了转基因拟南芥优良的除草剂抗性。
与现有技术相比，本发明的有益效果为：
(1)本发明的基于双分子脱氨酶互补的胞嘧啶碱基编辑系统相比传统胞嘧啶碱基编辑(如BE3系统)，降低了碱基编辑在基因组上的Cas9依赖型和Cas9非依赖型(随机型)脱靶，同时仍然保持了较为高效的碱基编辑中靶活性。
(2)本发明提供的基于双分子脱氨酶互补的腺嘌呤碱基编辑系统也展现出了在几乎所有代表性真核生物的普适性，在单子叶植物如水稻，双子叶植物如拟南芥，真菌如酵母，哺乳动物如人HEK293T细胞系均展现良好的腺嘌呤碱基编辑活性。
(3)本发明的基于双分子脱氨酶互补的碱基编辑系统的开发和应用，为生 物基因科学研究，作物遗传育种，尤其是人类遗传疾病的临床治疗提供了安全而有力的技术支撑。
图1为基于双分子脱氨酶互补的碱基编辑系统的工作原理图。
图2为用于在水稻原生质体中瞬时表达碱基编辑器的载体示意图。
图3：A为在水稻原生质体中使用R-loop方法评估多个碱基编辑器的中靶效率统计图(n＝3)；B为在水稻原生质体中使用R-loop方法评估多个碱基编辑器的脱靶效率统计图(n＝3)；C为图A和图B的综合统计图(n＝12)。
图4：A为4个水稻内源靶点的中靶编辑效率柱状图；B为比较split-AID10与两个传统碱基编辑器的Cas9依赖型脱靶编辑效率柱状图(n＝2)与脱靶位点信息表。
图5：A为split-AID10系统在3个拟南芥内源靶点的碱基编辑统计表；B为使用split-AID10系统编辑拟南芥内源ALS基因，从而赋予转基因T1代拟南芥对于咪唑啉酮类除草剂的抗性效果图和靶位点桑格测序图。
图6：A为酿酒酵母6个靶点的编辑效率统计图(n＝3)；B为为酿酒酵母中单细胞水平全基因组范围的脱靶编辑评估，图中柱状图的值代表了多个生物学重复的平均值(ns,统计不显著；****，p值小于0.0001)。
图7：A为人胚胎肾细胞系HEK293T中6个靶点的中靶编辑效率统计图(n＝3)；B为人胚胎肾细胞系HEK293T中6个靶点的脱靶编辑效率统计图(n＝3)；C为人胚胎肾细胞系HEK293T中图A和图B中所有靶点的中靶检测和脱靶检测编辑水平统计图(n＝18)；D为HEK293T细胞系中4个靶点的中靶编辑效率以及每个靶点对应的Cas9依赖型脱靶编辑水平统计图(n＝3)。
图8：A为酿酒酵母内源靶点Ade1-1每个碱基C的编辑效率统计图(n＝3)；B为酿酒酵母内源靶点Ade1-3每个碱基C的编辑效率统计图(n＝3)；C为酿酒酵母内源靶点Can1-1每个碱基C的编辑效率统计图(n＝3)；D为酿酒酵母 内源靶点Can1-9每个碱基C的编辑效率统计图(n＝3)。
图9：A为在拟南芥原生质体中，比较Split-ABE8e和Split-ABE8e-N7-C2系统与2个传统腺嘌呤碱基编辑系统靶向内源基因FLS2的编辑效率统计图(n＝2)；B和C为使用Split-ABE8e系统对转基因拟南芥(B)和转基因水稻(C)植株中各自3个内源基因的编辑情况统计图。
图10：A、B分别展示了酿酒酵母内源靶点Can1-5和Can1-9每个碱基A的编辑效率统计图(n＝3)；C、D分别展示了在人胚胎肾细胞系HEK293T靶点Site 1和Site 2的腺嘌呤碱基编辑情况。
为更好地说明本发明的目的、技术方案和优点，下面将结合具体实施例对本发明作进一步说明。本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。例如，本发明中使用的标准重组DNA和分子克隆技术为本领域技术人员熟知，并且在公开发表的文献中有更全面的描述
[7]。下述实施例中的实验方法，如无特殊说明，均为常规方法；所用的试验材料，如无特殊说明，均为可通过常规的商业途径购买获得。
如图1所示，本发明提供的基于双分子脱氨酶互补的碱基编辑系统主要由三部分组成：碱基编辑融合蛋白A，碱基编辑融合蛋白B以及向导RNA。其中，所述碱基编辑融合蛋白A和碱基编辑融合蛋白B共同构成双分子脱氨酶互补的碱基编辑器。所述融合蛋白A由第一nCas9多肽片段与第一脱氨酶多肽片段通过柔性连接肽融合而成。所述融合蛋白B由第二脱氨酶多肽片段与第二nCas9多肽片段通过柔性连接肽融合而成。所述第一，第二nCas9多肽片段可无缝融合成完整nCas9蛋白。
所述第一，第二脱氨酶多肽片段的脱氨酶选自同一核碱基脱氨酶，且其任一部分均不包含完整脱氨酶序列，也不具脱氨酶活性。在一些优选的实施方案中，如Split-AID10胞嘧啶碱基编辑系统(图2-7)，Split-BE3胞嘧啶碱基编辑系统(图6-7)，Split-A3A胞嘧啶碱基编辑系统(图8)，Split-A3B胞嘧啶碱 基编辑系统(图8)和Split-ABE8e腺嘌呤碱基编辑系统(图9-10)中，所述第一，第二脱氨酶多肽片段也可无缝融合成完整的相应核碱基脱氨酶。
在一些次优选的实施方案中，如Split-AID10-N5-C4胞嘧啶碱基编辑系统(图2)和Split-ABE8e-N7-C2腺嘌呤碱基编辑系统(图9)，其第一第二脱氨酶多肽片段有部分氨基酸序列的重叠，融合成的脱氨酶氨基酸序列长度超过原始所选核碱基脱氨酶。
其工作原理为：当向导RNA与碱基编辑融合蛋白A和B共表达时，向导RNA引导后两者组装成完整的碱基编辑器并，三者共同组成蛋白核酸复合体(RNPs)。随后RNPs由向导RNA靶向结合至目的基因位点处，导致所述目的基因序列中一至多个C碱基到T碱基的核苷酸取代(胞嘧啶碱基编辑系统)或一至多个A碱基到G碱基的核苷酸取代(腺嘌呤碱基编辑系统)。
本发明提供的实施例证明，前述优选的实施方案如Split-AID10胞嘧啶碱基编辑系统在植物如水稻，真菌如酵母，动物如人的HEK293T细胞系中均表现出接近背景水平的全基因组DNA脱靶，显著低于传统碱基编辑器如BE3(本发明也称“N-BE3”)，同时维持了较为高效的DNA中靶效率。前述优选的实施方案Split-BE3胞嘧啶碱基编辑系统在代表性真核生物酵母和人的HEK293T细胞系中也表现出接近背景水平的全基因组DNA脱靶，显著低于传统碱基编辑器BE3，同时维持了较为高效的DNA中靶效率。前述优选的Split-ABE8e腺嘌呤碱基编辑系统也展示出了广泛的物种适应性，从单细胞真核生物酵母，到高等植物拟南芥和水稻，乃至人的HEK293T细胞系中均能实现对真核生物内源靶点的有效腺嘌呤碱基编辑。
实施例1在单子叶模式植物水稻中评估基于双分子脱氨酶互补的胞嘧啶碱基编辑系统的中靶效率和脱靶效率。
1、实验材料：本实施例所使用野生型水稻品种为粳稻中花11(Oryza sativa L.ssp.japonica ZH11)。
2、碱基编辑相关水稻原生质体瞬时表达载体的构建
碱基编辑器“N-BE3”(或称为“BE3”)的表达载体pHBT-rAPOBEC1-nCas9-UGI由本实验室自主构建，其所用启动子为玉米ZmUbi-1启动子，所用终止子为NOS终止子。
基于双分子脱氨酶互补的胞嘧啶碱基编辑器“Split-AID10”由“Split-AID10-N”表达载体和“Split-AID10-C”表达载体共同构成；由上海生工生物技术有限公司(Sangon Biotech.Co.,Ltd.)商业化合成编码氨基酸序列如SEQ ID NO:15(Split-AID10-N)所述多肽片段的多核苷酸序列，随后将该产物经简单酶切连接融合至前述pHBT载体获得表达载体pHBT-Split-AID10-N；“Split-AID10-C”表达载体的构建：pHBT-Split-AID10-C载体构建过程与pHBT-Split-AID10-N基本一致，其包含编码氨基酸序列如SEQ ID NO:16(Split-AID10-C)所述多肽片段的多核苷酸序列。
基于双分子脱氨酶互补的胞嘧啶碱基编辑器“Split-AID10-N5-C4”由表达载体pHBT-Split-AID10-N5和表达载体pHBT-Split-AID10-C4共同构成，构建方法同“Split-AID10”，其中Split-AID10-N5的氨基酸序列如SEQ ID NO:17，Split-AID10-C4的氨基酸序列如SEQ ID NO:18。
nSaCas9表达载体的构建：植物密码子优化的SaCas9的多核苷酸序列来自德国Puchta Holger教授的馈赠(已公开论文
[8])。使用购自南京诺赞生物技术有限公司(Vazyme Biotech Co.,Ltd)的定点突变试剂盒(Mut Express II Fast Mutagenesis KitV2，#C214-01)，将前述SaCas9编码氨基酸序列中的第10位天冬氨酸突变为丙氨酸，获得编码nSaCas9(D10A)的多核苷酸序列。最后通过简单酶切连接方法构建表达载体pHBT-nSaCas9。
3、水稻向导RNA瞬时表达载体的构建
参照已公开发表的论文中阐述的方法
[9,10]，并基于pUC119-OsU6apro-sgRNA
[10]构建8个向导RNA表达载体pUC119-OsAAT1-sgRNA、pUC119-OsCDC48-sgRNA、pUC119-OsNAL1-sgRNA、pUC119-OsPDS1-sgRNA、pUC119-OsCDC48-Sa-gRNA、pUC119-OsNRT1.1B-Sa-gRNA、pUC119-OsDEP1-Sa-gRNA和 pUC119-OsAAT1-Sa-gRNA。向导RNA所包含的靶序列信息如表1所示。
表1
4、水稻原生质体的制备及转染
水稻原生质体制备：
1)12小时光照(32℃)/12小时黑暗(28℃)，200μmol·m
-2·s
-1光照，70％湿度，土培或者无菌组培8-10天龄水稻幼苗；
2)配制酶解液(1.5％纤维素酶R10，0.4％果胶酶R10，0.4M甘露醇，20mM MES pH 5.7，20mM KCl，10mM CaCl
2和0.1％BSA)15mL，并用针筒和0.45μm孔径的滤膜过滤，加入至直径10cm的细胞培养皿中；
3)选取约200颗生长健康、颜色嫩绿的水稻苗，用刀片把叶鞘切成0.5-1mm的茎段；
4)将茎段放入前述酶解液中，注意用接种环将各个茎段分散，避免粘连，并使其完全浸没于酶解液中，置于50-60rpm转速的水平摇床上，避光酶解3小时；
5)酶解完成后，加入10mL W5(154mM NaCl，125mM CaCl
2，5mM KCl和2mM MES pH 5.7)溶液，用适当的力度摇晃培养皿，使原生质体释放于溶液中，此时可以看到溶液变淡绿，用孔径45μm的尼龙膜过滤酶解产物，收集滤液至圆底离心管中；
6)在水平离心机中以150g离心5分钟，然后用真空泵吸去尽可能多的上清液，淡绿色原生质体沉于圆底管底部；
7)缓慢加入10mL W5溶液，轻轻摇晃圆底管以重悬细胞，随后于冰上静置0.5-1小时；
8)冰上静置后，以150g水平离心3分钟，吸去上清，缓慢加入2mL MMg(0.4M甘露醇，15mM MgCl
2和4mM MES pH5.7)溶液并轻摇圆底管以重悬原生质体，用血球计数板在显微镜下确定细胞浓度，再次加入适量MMg溶液调整细胞浓度至2×10
6个/mL。
水稻原生质体转染：
向2mL圆底管中依次加入300μL前述调整好细胞浓度的水稻原生质体、30μL(66μg)质粒和330μL PEG(40％PEG4000(v/v)，0.2M甘露醇和0.1M CaCl
2)，反应液轻柔地充分混匀，避光静置15分钟，随后加入1.2mL W5溶液， 混匀，从而终止转染。水平离心机200g离心5分钟，吸去上清，加入150μL W5溶液重悬，并转移至1mLWI溶液(0.5M甘露醇，20mM KCl和4mM MES pH 5.7)中。室温黑暗培养48小时，水平离心机250g离心5分钟，吸上清，淡黄色原生质体细胞聚集于管底，之后迅速冻于液氮，于-80℃冰箱保存。
5、水稻原生质体基因组中靶序列的深度测序和碱基替换分析
1)使用购自天根生化科技(北京)有限公司(TIANGEN Biotech.Co.,Ltd.)的快捷型植物基因组DNA提取系统(DNAquick Plant System,#4992710)提取水稻原生质体DNA；
2)设计带有barcode标签序列的第一轮特异性引物扩增靶序列上下游200bp左右的片段，在第一轮PCR产物的基础上使用通用建库引物进行第二轮建库PCR引物信息列于表2；
3)按等摩尔比混合前述第二轮建库PCR产物，纯化后送往北京诺禾致源科技股份有限公司(Novogene Co.,Ltd.)进行扩增子测序。使用NovaSeq平台进行双端PE150测序；
4)在公共数据分析平台上(https://usegalaxy.org/)
[11]根据barcode拆分前述测序数据，最后使用CRISPResso2
[12]或BE-Analyzer
[13]网站进行靶序列突变分析，用Excel呈现靶位点碱基替换情况。
表2引物信息
6、基于正交R-loop的高通量碱基编辑器特异性检测方法
参照已公开发表的论文中阐述的方法
[14]。本实施例中选择了8个水稻内源靶点，包括4个SpCas9的向导RNA靶向的中靶位点(OsAAT1，OsCDC48，OsNAL1与OsPDS1)，与4个nSaCas9的向导RNA靶向的脱靶位点(OsCDC48,OsNRT1.1B,OsDEP1,OsAAT1)。为了模拟碱基编辑器在细胞内的真实工作情况，将前述靶位点按照SpCas9与SaCas9一一对应的方式随机分组，获得Sp-OsAAT1与Sa-OsCDC48,Sp-OsCDC48与Sa-OsNRT1.1B,Sp-OsNAL1与Sa-OsDEP1和Sp-OsPDS1与Sa-OsAAT1共4个组合。在每个组合的试验中，每个水稻原生质体的处理组须等量地转染碱基编辑器质粒(特别地，Split-AID10和Split-AID10-N5-C4碱基编辑器则须转染等量pHBT-Split-AID10-N(N5)质粒和pHBT-Split-AID10-C(C4)质粒)，SpCas9向导RNA质粒(如pUC119-OsAAT1-sgRNA)，nSaCas9质粒(pHBT-nSaCas9)以及nSaCas9向导RNA质粒(如pUC119-OsCDC48-Sa-gRNA)。针对每个组合试验中的每个处理组，同时进行SpCas9靶点(中靶)和nSaCas9靶点(脱靶)的深度测序和分析。
7、水稻遗传转化载体的构建和水稻的遗传转化
1)碱基编辑器BE3的遗传转化载体为pH-nCas9-PBE(addgene:#98163)，其中所用启动子为ZmUbi-1启动子，所用终止子为E9终止子。靶向编辑目的基因OsSWEET11/13/14：商业化合成3个向导RNA的表达框OsU6apro-OsSWEET14-OsU6bpro-OsSWEET13-OsU6apro-OsSWEET11，通过简单酶切连接融合进前述pH-nCas9-PBE载体中(靶序列信息列于表1)。靶向编辑目的基因OsSLR1：商业化合成单个向导RNA的表达框OsU6apro-OsSLR1,通过简单酶切连接融合进前述pH-nCas9-PBE载体中(靶序列信息列于表1)；
2)Split-AID10碱基编辑器的遗传转化载体,第一步，以pHBT-Split-AID10-N载体为模板，经简单PCR与酶切连接获得pH-Split-AID10-N载体。第二步，首先从水稻中花11基因组上经PCR获得OsUBQ2启动子序列，再以载体pHBT-Split-AID10-C和pH-nCas9-PBE为模板通过简单重叠PCR获得Split-AID10-C-E9term多核苷酸序列，通过简单PCR将前两者融合获得Split-AID10-N的表达框OsUBQ2pro-Split-AID10-C-E9term多核苷酸序列。最后通过简单酶切连接将前述Split-AID10-N的表达框融合进pH-Split-AID10-N载体的E9终止子之后，获得拥有2个完整表达框的pH-Split-AID10-N-C双元载体。靶向编辑目的基因OsSWEET11/13/14和OsSLR1的2个双元载体如前所述克隆至pH-PIGS-AID10中。
3)水稻遗传转化：将上述载体利用冻融转化法转入农杆菌(Agrobacterium tumefaciens)株系EHA105感受态细胞，后续委托武汉伯远生物科技有限公司(BioRun Co.,Ltd.)进行水稻的遗传转化工作。
8、实验结果
首先，采用前述基于正交R-loop的方法(图3A，3B)在水稻原生质体中评估2种基于双分子脱氨酶互补的胞嘧啶碱基编辑系统Split-AID10和Split-AID10-N5C4的中靶与脱靶效率，同时选择传统碱基编辑器N-BE3作对照(图2)。中靶编辑方面，分别针对4个SpCas9向导RNA靶点所处的基因组区域进行深度测序，分析结果显示，碱基编辑器Split-AID10和Split-AID10-N5C4均能对水稻内源靶位点进行有效的碱基编辑，平均C到T碱基替换效率约为4.6％和6.6％，大于传统碱基编辑器N-BE3(BE3)的3.3％(图3A，3C)。脱靶编 辑方面，分别针对4个nSaCas9向导RNA靶点进行深度测序分析，结果显示Split-AID10和Split-AID10-N5C4在4个位点的平均脱靶编辑效率(0.05％和0.8％)均显著低于传统碱基编辑器BE3(1.6％)。尤其是碱基编辑器Split-AID10，其平均脱靶效率仅为0.05％，与空白对照的0.02％基本一致(图3B，3C)。
随后，将前述原生质体中表现最优的Split-AID10碱基编辑器与向导RNA融合构建遗传转化载体进行水稻的遗传转化，在水稻转基因植株中评估该碱基编辑器，同时选择传统碱基编辑器N-BE3作对照。获得转基因植株后，针对不同靶基因位点设计特异性引物进行PCR，将产物送擎科生物技术有限公司(Tsingke Biotech.Co.,Ltd.)进行桑格测序。针对4个靶点的分析结果显示，碱基编辑器Split-AID10在转基因植株中也成功进行碱基编辑，有效编辑窗口为靶序列的C
5位到C
13位(以PAM为21-23位)，不同位置的胞嘧啶碱基替换效率从3％到55.2％(图3A)。最后，我们使用在线分析工具CRISPR-GE(http://skl.scau.edu.cn/)分析了全部4个靶点可能的Cas9依赖型脱靶情况。以该工具脱靶得分>0.7为标准，仅在中靶位点OsSWEET13中寻找到2个可能的脱靶位点(图3B)。随后针对3种碱基编辑器BE3和Split-AID10所有在该位点成功编辑的转基因水稻植株共38株进行2个脱靶位点的桑格测序。如图3B所示，Split-AID10仅在平均50％的已编辑植株发现前述Cas9依赖型的DNA脱靶情况，显著小于BE3的100％。
综上所述，在水稻中2种基于双分子脱氨酶互补的胞嘧啶碱基编辑系统Split-AID10和Split-AID10-N5C4均能进行有效碱基编辑，且相比传统碱基编辑器如BE3，显著降低了碱基编辑的全基因组随机脱靶概率(同“Cas9非依赖型脱靶”)，同时维持了较高的中靶编辑效率。进一步，Split-AID10在转基因水稻植株水平也展现了良好的碱基编辑中靶效率，同时发现其降低Cas9依赖型脱靶发生机率。
实施例2使用基于双分子脱氨酶互补的胞嘧啶碱基编辑系统Split-AID10也可在转基因拟南芥植株中实现高效碱基编辑。
1、实验材料
本实施例所用野生型拟南芥为Col-0生态型(Arabidopsis thaliana Col-0)。
2、拟南芥遗传转化
本实施例所用拟南芥遗传转化载体的构建参考公开发表文章
[15]和实施例1所述方法，针对2个不同靶点分别构建各自遗传转化载体(向导RNA靶序列信息见表1)。将前述载体分别通过电击转化农杆菌(Agrobacterium tumefaciens)株系GV3101，利用花粉管导入法转化拟南芥植株。具体地，将含有目标载体的GV3101菌液，按1:100的比例接种于含卡那霉素(50mg/L)的液体LB培养基中，在28℃摇床中以转速220rpm培养2天。5000g收集菌体，弃去培养基，加入含0.05％Silwet L77的5％蔗糖溶液重悬。取已开花的拟南芥植株，倒置并使花序完全浸入农杆菌菌液中，轻轻搅动约10秒后取出，置于湿润黑暗环境中1天后，转移至正常生长环境，直至收获成熟种子。
3、转基因阳性苗筛选及基因型鉴定
将前述获得的成熟种子在无菌环境下播撒于含有50mg/L的潮霉素B(hygromycin B)的无菌1/2MS固体培养基中。10天左右，将生根的潮霉素阳性苗移至土壤中恢复培养。半个月后，使用购自购自天根生化科技(北京)有限公司的快捷型植物基因组DNA提取系统提取阳性苗的叶片基因组。设计靶基因特异性引物进行PCR，并送擎科公司进行桑格测序。
4、实验结果
如图4A所示，对2个独立内源靶点AtALS和AteTM166的转基因拟南芥基因型鉴定结果显示，Split-AID10在转基因拟南芥植株中可以成功进行有效的C到T碱基编辑，平均编辑效率为7.7％。如图4B所示，在使用Split-AID10编辑拟南芥内源ALS基因所获得的T1代转基因植株中，其靶位点上发生预想的G
13到A
13的碱基替换，从而导致其内源ALS基因编码序列上第122位丙氨酸到苏氨酸(A122T)的氨基酸取代。对2月龄的前述所有T1代转基因拟南芥植株(不论是否发生碱基编辑)进行咪唑啉酮类除草剂(Shandong Cynda Chemical Co.,Ltd.)的喷施，1月后发现存活植株均包含内源ALS基因上G
13到A
13的碱基替换，说明使用Split-AID10碱基编辑系统靶向编辑拟南芥内源基因ALS赋予了 转基因拟南芥植株对咪唑啉酮类除草剂的抗性。本实施例证明Split-AID10碱基编辑系统在转基因拟南芥植株中可进行有效地C到T碱基编辑，并且能赋予拟南芥优良的农艺性状(如除草剂抗性)。
实施例3基于双分子脱氨酶互补的胞嘧啶碱基编辑系统Split-AID10和Split-BE3在单细胞真核生物酵母中也展现出良好的中靶编辑和极低的全基因组随机脱靶。
1、实验材料
本实施例所用为酿酒酵母BY4741株系(Saccharomyces cerevisiae BY4741)。
2、碱基编辑相关酵母表达载体构建
碱基编辑相关表达载体构建参考公开发表文章
[16]，获得表达载体pGAL1-rAPOBEC1-nCas9-UGI(N-BE3),pGAL1-Split-AID10和pGAL1-Split-BE3。其中，pGAL1-Split-AID10表达载体所包含的Split-AID10-N和Split-AID10-N氨基酸序列如前所述，pGAL1-Split-BE3表达载体所包含的Split-BE3-N的氨基酸序列如SEQ ID NO：8所示，Split-BE3-C的氨基酸序列如SEQ ID NO：9所示。酵母U6启动子直接从基因组中PCR扩增获得，然后通过重叠PCR的策略组装向导RNA完整表达原件，完成一系列向导RNA的载体构建pGAL1-yGFP-SNR35p-sgRNAs，酵母中向导RNA靶点信息见表3。
表3酵母细胞中向导RNA靶点信息
| 靶基因 | 靶序列(5’-3’) |
| PolyC-1 | CCCCCCCCATGTTCCGAGAT CGG |
| PolyC-2 | TCCCCCCCCTCAATTCCAGC AGG |
| PolyC-3 | ATCAGCCCCCCCCCAAGGAA AGG |
| PolyC-4 | GAACAGCTGAACCCCCCCAA TGG |
| PolyC-5 | CATTAAAGCAACCCCCCATA GGG |
| Can1-5 | TCCAATAACGGAATCCAACT GGG |
| Can1-9 | CACAAACACACCACAGACGT GGG |
3、酵母遗传转化和表达
所采用的酿酒酵母遗传转化参考公开发表文章
[17]。通过常规醋酸锂LiOAC化学转化法将前述载体转入酿酒酵母BY4741株系中，利用质粒上的营养缺陷基因筛选阳性转化子。具体地，从营养缺陷平板上挑取含有目标质粒的阳性转化克隆到液体缺陷培养基中，在2％葡萄糖碳源的培养基培养两天至饱和，然后按照1:1000的稀释比例接种于2％棉子糖碳源培养基培养两天至饱和，最后按照1:10000的稀释比例接种于1％半乳糖碳源培养基培养两到三天至饱和，收集适量的菌液进行后续基因组提取。
4、酵母细胞中靶序列的深度测序及全基因组深度测序
1)对于中靶序列的深度测序，参考公开发表文章
[18]进行基因组提取和靶点的扩增子建库。
2)对于全基因的深度测序，取适量半乳糖诱导的饱和菌液进行平板划线，待克隆长成后，挑取10～20个单克隆进行一代测序，鉴定对应靶点有没有编辑；将靶点已编辑的克隆接种到3mL的YPDA液体培养基中并摇菌至饱和，然后用商业化的试剂盒(HiPure Yeast DNA Kit,#D3147,Guangzhou Magen Biotech.Co.,Ltd.)抽提酵母菌的基因组DNA，再利用商业化的二代测序建库试剂盒完成文库构建(VAHTS Universal DNA Library Prep Kit for Illumina,#ND607,Vazyme Biotech.Co.,Ltd.)，最后送测序公司进行双端PE150高通量测序，要求每个克隆测序覆盖深度超过100×，大约每个基因组需1G数据。
5、实验结果
如图6A所示，针对酵母基因组内源6个靶点的分析结果显示，碱基编辑器N-BE3平均编辑效率为6.5％～94.9％，有效编辑窗口为靶序列的C
3位到C
10位；碱基编辑器Split-AID10平均编辑效率为56.9％～97.3％，有效编辑窗口为靶序列的C
5位到C
15位；碱基编辑器Split-BE3平均编辑效率为57.8％～96.8％，有效编辑窗口为靶序列的C
5位到C
14位。此外，与经典的碱基编辑器N端融合策略N-BE3相比，Split-AID10和Split-BE3的编辑窗口都发生了偏移，更靠近PAM 识别区域。
如图6B所示，无论是使用脱氨酶AID10的Split-AID10系统还是使用脱氨酶rAPOBEC1的Split-BE3系统，其均能够显著降低全基因范围的脱靶效应。尽管酵母基因组非常小(只有10Mb左右)，碱基编辑器N-BE3还是导致了全基因组范围内平均可以检测到100个左右脱靶编辑事件，而Split-AID10和Split-BE3都能够极大地降低脱靶效应，平均只能检测不到10个的脱靶编辑，与单分子碱基编辑器对照组Split-AID10-N/Split-AID10-C和Split-BE3-N/Split-BE3-C数目相当，接近本底水平。
本实施例证明双分子脱氨酶互补的碱基编辑系统在真核细胞酿酒酵母中具有高效的编辑活性，同时，能够显著地降低全基因组范围的脱靶效应，具有非常高的安全性能。
实施例4基于双分子胞苷脱氨酶互补的Split-AID10和Split-BE3系统在人胚胎肾细胞系293T中也展现出良好的中靶编辑和极低的全基因组随机脱靶。
1、实验材料
人胚胎肾细胞系293T(HEK293T)来自美国模式培养物集存库(ATCC)。
2、人碱基编辑相关载体的构建
参考已公开发表的文章
[19]，核心载体整合了CMV启动子和人源U6启动子，载体骨架为pX330(Addgene#42230)，然后构建相关碱基编辑器pCMV-rAPOBEC1-nCas9-UGI(N-BE3),pCMV-Split-AID10和pCMV-Split-BE3表达载体,其中pCMV-Split-AID10表达载体所包含的Split-AID10-N和Split-AID10-C氨基酸序列如前所述，pCMV-Split-BE3表达载体包含的Split-BE3-N和Split-BE3-C氨基酸序列也如前所述；本实施例对向导RNA表达载体做了如下改造，在CMV下游插入红色荧光蛋白mScarlet，用于指示细胞转染的效率，同时在U6和gRNA Scaffold之间通过BbsI酶切消化，插入对应靶点的向导RNA退火双链分子，完成向导RNA表达元件的构建，HEK293T细胞中向导RNA靶点信息见下表4。
表4人基因组中向导RNA靶序列信息
3、细胞培养和转染
HEK293T细胞为人胚胎肾细胞，贴壁生长，采用DMEM高糖培养基和10％胎牛血清培养，置于37℃和5％二氧化碳的培养箱培养。进行碱基编辑器质粒转染前一天，铺合适数目的细胞于48孔板中，第二天采用常规的脂质体进行细胞转染，表达48-72小时后，收集细胞并进行基因组提取。
4、HEK293T细胞中靶序列的深度测序和分析
为了更好的评估互补的双分子脱氨酶碱基编辑器的性能，本实施例设计了6个匹配的靶点，同时进行碱基编辑器的中靶效果和脱靶效果检测。具体实施如下：以SaCas9搜索基因组合适的靶位点，然后筛选符合SpCas9相邻间隔原基序PAM的要求，获得重叠的靶序列，从而针对同一个位点进行靶向编辑的效率评估和正交的R-Loop检测。其中，R-Loop正交检测使用了pX601(Addgene#61591)作为表达载体，为了提高检测效率，表达载体pX601有适当改造，包 括使用nSaCas9切口酶和增加了尿嘧啶糖基化酶抑制功能结构域UGI，正交检测的靶位点信息详见表4。
深度测序建库采用两步法策略：1)首先设计靶点特异性的结合引物进行捕获，并且在引物末端带上Illumina Nextera接头的部分序列；2)然后以第一步反应的PCR产物为模板，进行完整文库的扩增，包括8个碱基的样品条形码和P5/P7的完整序列。最后，两轮PCR扩增的产物进行纯化、浓度测定和文库均一化混匀等一系列常规操作，送至苏州安升达公司(Genewiz Inc.,sz)进行高通量测序。
扩增子深度测序结果分析主要包含文库拆分和编辑效率计算两个部分。文库拆分利用Illumina官方的bcl2fastq软件进行，将原始下机的BCL数据根据混合样品的条形码表格挨个拆分成独立的FASTQ格式文件，包括读长1(Read1)和读长2(Read2)；然后针对每个靶点单独进行CRISPResso2
[12]分析，获得靶点附近碱基替换的矩阵，最后完成编辑效率的计算。
5、Cas9依赖性的脱靶检测
本实施例还针对4个靶点进行了Cas9蛋白依赖性的脱靶检测，通过预测软件Cas-OFFinder针对每个靶点序列进行小于6的容错搜索，从人类全基因组范围内寻找Cas9蛋白依赖性脱靶位置；然后再根据CRIPSR/Cas9系统的核心种子区域进行筛选，保留靠近PAM区域5个碱基长度没有错配的潜在脱靶位点序列；最后根据已发表的大量文献和方法，设计一套相对简易的打分算法，对潜在的脱靶位点序列特征进行分析，进一步筛选获得打分较高的位点，设计特异性的捕获引物进行后续扩增子的建库和测序。
6、实验结果
实验结果如图7所示，由图7A可知，基于胞苷脱氨酶rAPOBEC1的Split-BE3系统和基于胞苷脱氨酶AID10的Split-AID10系统在HEK293T细胞能够成功实现靶序列C到T的碱基替换，编辑效率与常用的N-BE3碱基编辑器相当，平均编辑效率在50％左右。由图7B～D可知，Split-AID10和Split-BE3不仅能够减少Cas9依赖性的脱靶编辑，还能够显著地降低Cas9非依赖性的脱靶风险。本实施例证明双分子脱氨酶互补的碱基编辑系统在人源HEK293T细胞中具有高效的编 辑活性，同时，不仅能够减少Cas9依赖性的脱靶编辑，而且能够显著地降低全基因组范围的脱靶效应，表现出优异的安全性能，具有广阔的实际应用前景。
实施例5 Split-A3A和Split-A3B系统在真核生物中具有良好的基因编辑能力。
1、碱基编辑相关酵母表达载体构建
参照实施例3所述方法，Split-A3A系统的表达载体为pGAL1-Split-A3A-N和pGAL1-Split-A3A-C；编码Split-A3A-N的氨基酸序列如SEQ ID NO：10所示，编码Split-A3A-C的氨基酸序列如SEQ ID NO：11所示；Split-A3B系统的表达载体为pGAL1-Split-A3B-N和pGAL1-Split-A3B-C，编码Split-A3B-N的氨基酸序列如SEQ ID NO：12所示，编码Split-A3B-C的氨基酸序列如SEQ ID NO：13所示。
2、实验结果
如图8A-8D所示，基于胞苷脱氨酶A3A的Split-A3A系统和基于胞苷脱氨酶A3B的Split-A3B系统在代表性真核生物酵母中成功实现靶序列C到T的碱基替换。Split-A3A系统的编辑窗口为C
1到C
15，在编辑窗口内不同位置的C碱基的编辑效率为22.1％～84.7％；Split-A3B系统的编辑窗口为C
5到C
15在编辑窗口内不同位置的C碱基的编辑效率为9.5％～95.5％。
实施例6基于双分子脱氨酶互补的腺嘌呤碱基编辑系统Split-ABE8e和Split-ABE8e-N7-C2可在植物中实现高效腺嘌呤碱基编辑。
1、实验材料
本实施例中水稻材料如实施例1相同，拟南芥材料如实施例2相同。
2、拟南芥原生质体瞬时转化载体的构建
传统腺嘌呤碱基编辑器ABE7.10和ABE8e的拟南芥瞬时表达载体为pHBT-ABE7.10和pHBT-ABE8e，其中所用启动子为AtUBQ10启动子，终止子为NOS终止子。前述载体均为本实验室构建，已公开发表
[15]；Split-ABE8e系 统和Split-ABE8e-N7-C2系统表达载体的构建参照实施例1所述Split-AID10系统以及Split-AID10-N5-C4系统的构建方法，在pHBT-PIGS-ABE8e载体的基础上获得Split-ABE8e系统的pHBT-Split-ABE8e-N载体和pHBT-Split-ABE8e-C载体以及Split-ABE8e-N7-C2系统的pHBT-Split-ABE8e-N7载体和pHBT-Split-ABE8e-C2载体。其中，编码Split-ABE8e-N的氨基酸序列如SEQ NO:29，编码Split-ABE8e-C的氨基酸序列如SEQ NO:30，编码Split-ABE8e-N7的氨基酸序列如SEQ NO:31，编码Split-ABE8e-C2的氨基酸序列如SEQ NO:32；适用于拟南芥的向导RNA瞬时表达载体基于Li,Z.et al.2019.Currentprotocols in molecular biology,https://doi.org/10.1002/cpmb.89公开发表文章所述pUC119-AtU6-26pro-sgRNA)载体，构建pUC119-AtU6-26pro-AtFLS2-sgRNA表达载体(靶序列参见表1)。
3、拟南芥原生质体基因组中靶序列的深度测序和碱基替换分析
本实施例的拟南芥原生质体基因组中靶序列的深度测序和碱基替换分析与实施例1相同。
4、植物遗传转化载体的构建
参考实施例1所述Spit-AID10水稻遗传转化载体的构建方法构建本实施例Split-ABE8e系统适用于水稻的遗传转化载体pH-Split-ABE8e-N-C；在此基础上将商业化合成的单向导RNA表达框AtU6-26pro-AtALS/AtPDS3/AtBAK1-sgRNA核酸片段通过简单酶切连接各自融入前述pH-Split-ABE8e-N-C载体中，获得本实施例中靶向AtALS或AtPDS3或AtBAK1的水稻遗传转化载体(向导RNA靶序列信息见表1)。
参考实施例2所述Spit-AID10拟南芥遗传转化载体的构建方法构建本实施例Split-ABE8e系统适用于拟南芥的遗传转化载体pH-EC-Split-ABE8e-N-C；在此基础上将商业化合成的单向导RNA表达框AtU6-26pro-OsACC/OsNRT1.1B/OsDEP1-sgRNA核酸片段通过简单酶切连接各自融入前述pH-EC-Split-ABE8e-N-C载体中，获得本实施例中靶向OsACC或OsNRT1.1B或OsDEP1的拟南芥遗传转化载体(向导RNA靶序列信息见表1)。
5、植物遗传转化
水稻遗传转化方法参照实施例1所述，拟南芥遗传转化方法参照实施例2所述。
6、实验结果
为快速验证基于双分子脱氨酶互补的腺嘌呤碱基编辑系统Split-ABE8e和Split-ABE8e-N7-C2的可行性，将表达Split-ABE8e系统所必须的两组分Split-ABE8e-N表达载体和Split-ABE8e-C表达载体与靶向AtABI3-2位点的向导RNA表达载体共同转染拟南芥原生质体，同时也将表达Split-ABE8e-N7-C2系统所必须的两组分Split-ABE8e-N7表达载体和Split-ABE8e-C2表达载体与前述向导RNA表达载体共同转染另一组原生质体，除此之外还设置了传统腺嘌呤碱基编辑器ABE7.10和ABE8e的对照组。48小时后，提取原生质体基因组DNA并对靶位点AtFLS2进行深度测序。结果如图9A所示，使用2种新型腺嘌呤碱基编辑系统Split-ABE8e和Split-ABE8e-N7-C2均能有效编辑拟南芥内源靶点AtFLS2，对该靶点7位的A碱基的平均编辑效率为3.3％和3.2％，与传统碱基编辑器ABE8e的3.2％编辑效率相似，同时高于传统ABE7.10的0.3％。
为进一步确认该新型编辑系统的有效性，拟在两种代表性植物的转基因植株水平评估该碱基编辑系统的腺嘌呤碱基编辑效率。如图9B所示，在转基因拟南芥植株中，使用Spit-ABE8e系统分别靶向内源基因AtALS，AtPDS3和AtBAK1，腺嘌呤碱基编辑效率在3.1％到35.2％之间，平均为16.6％。如图9C所示，在转基因水稻植株中，使用Spit-ABE8e系统分别靶向内源基因OsACC，OsDEP1和OsNRT1.1B，腺嘌呤碱基编辑效率在55.1％到53.8％之间，平均为54.7％。
本实施例提供的数据证明，基于双分子脱氨酶互补的腺嘌呤碱基编辑系统Split-ABE8e和Split-ABE8e-N7-C2在植物中可以进行有效地腺嘌呤碱基编辑。特别是Split-ABE8e系统在单子叶代表性植物水稻和双子叶代表性植物拟南芥的转基因植株中均能对内源靶基因进行高效腺嘌呤碱基编辑，揭示了该系统在植物中广泛的适用性。
实施例7 Split-ABE8e系统可在酵母和人胚胎肾细胞系HEK293T中实现有效腺嘌呤碱基编辑。
1、实验材料
本实施在酵母中的实验材料与方法参考实施例3；在HEK293T细胞系中的实验材料与方法参考实施例4。
2、Split-ABE8e系统在酵母中的表达载体为pGAL1-Split-ABE8e-N和pGAL1-Split-ABE8e-C，其余对照载体为pGAL1-nSpCas9、pGAL1-N-ABE7.10和pGAL1-N-ABE8e；Split-ABE8e系统在HEK293T细胞系中的表达载体为pCMV-Split-ABE8e-N和pCMV-Split-ABE8e-C；前述酵母和人Split-ABE8e表达载体所包含的Split-ABE8e-N和Split-ABE8e-C的编码氨基酸序列如前所述；酵母和人细胞中向导RNA表达载体如实施例3，4所述，向导RNA靶序列信息见表3和表4。
3、实验结果
首先，将Split-ABE8e系统与靶向酵母内源Can1-8位点的向导RNA共同导入酵母细胞中，对靶点附近进行深度测序，结果如图10A所示，Split-ABE8e系统在酵母中成功对Can1-8位点进行了腺嘌呤碱基编辑，编辑窗口为A
4-A
13，A碱基到G碱基的替换效率从25％-82％。图10B展示Split-ABE8e系统在酵母细胞中另一个内源位点Can1-9的成功编辑情况。随后将Split-ABE8e系统与靶向内源HEK Site 1位点的向导RNA共同转染进HEK293T细胞系中，表达3天后，提取细胞基因组，使用引物F和R对HEK Site 1进行简单PCR，所得产物进行桑格测序。结果如图10C所示，Split-ABE8e系统在HEK293T细胞系中成功实现了腺嘌呤碱基编辑，将内源靶位点HEK Site 1中A
5替换为G
5。图10D展示Split-ABE8e系统在HEK293T细胞系中另一个内源位点HEK Site 2的成功编辑情况。
本实施例所展示的实验结果证明，Split-ABE8e系统在代表性单细胞生物如酵母和哺乳动物如人胚胎肾细胞系293T中均能实现对内源靶点有效地A到G碱基替换，进一步证实了该系统广泛的适应性。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围加以限制。尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
参考文献：
[1]WU J,ZENG Q,WANG Q,et al.SNP-based pool genotyping and haplotype analysis accelerate fine-mapping of the wheat genomic region containing stripe rust resistance gene Yr26[J].TAG Theoretical and applied genetics Theoretische und angewandte Genetik,2018,131(7):1481-96.
[2]LANDRUM M J,LEE J M,BENSON M,et al.ClinVar:public archive of interpretations of clinically relevant variants[J].Nucleic Acids Res,2016,44(D1):D862-8.
[3]ANZALONE A V,KOBLAN L W,LIU D R.Genome editing with CRISPR-Cas nucleases,base editors,transposases and prime editors[J].Nat Biotechnol,2020,38(7):824-44.
[4]KOMOR A C,KIM Y B,PACKER M S,et al.Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage[J].Nature,2016,533(7603):420-4.
[5]JIN S,ZONG Y,GAO Q,et al.Cytosine,but not adenine,base editors induce genome-wide off-target mutations in rice[J].Science,2019,364(6437):292-5.
[6]ZUO E,SUN Y,WEI W,et al.Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos[J].Science,2019,364(6437):289-92.
[7]J.R S,MANIATIS T,SAMBROOK J,et al.Molecular Cloning-A Laboratory Manual[J].2001,
[8]STEINERT J,SCHIML S,FAUSER F,et al.Highly efficient heritable plant genome engineering using Cas9 orthologues from Streptococcus thermophilus and Staphylococcus aureus[J].The Plant journal:for cell and molecular biology,2015,84(6):1295-305.
[9]LI J F,NORVILLE J E,AACH J,et al.Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9[J].Nat Biotechnol,2013,31(8):688-91.
[10]LI Z,WANG F,LI J F.Targeted Transcriptional Activation in Plants Using a Potent Dead Cas9-Derived Synthetic Gene Activator[J].Curr Protoc Mol Biol,2019,127(1):e89.
[11]AFGAN E,BAKER D,BATUT B,et al.The Galaxy platform for accessible,reproducible and collaborative biomedical analyses:2018 update[J].Nucleic Acids Res,2018,46(W1):W537-w44.
[12]CLEMENT K,REES H,CANVER M C,et al.CRISPResso2 provides accurate and rapid genome editing sequence analysis[J].Nat Biotechnol,2019,37(3):224-6.
[13]HWANG G H,PARK J,LIM K,et al.Web-based design and analysis tools for CRISPR base editing[J].BMC bioinformatics,2018,19(1):542.
[14]JIN S,FEI H,ZHU Z,et al.Rationally Designed APOBEC3B Cytosine Base Editors with Improved Specificity[J].Mol Cell,2020,79(5):728-40 e6.
[15]XIONG X,LI Z,LIANG J,et al.A cytosine base editor toolkit with varying activity windows and target scopes for versatile gene manipulation in plants[J].Nucleic Acids Res,2022,50(6):3565-80.
[16]LIU K,DENG S,YE C,et al.Mapping single-cell-resolution cell phylogeny reveals cell population dynamics during organ development[J].Nat Methods,2021,18(12):1506-14.
[17]CHEN P,WANG D,CHEN H,et al.The nonessentiality ofessential genes in yeast provides therapeutic insights into a human disease[J].Genome research,2016,26(10):1355-62.
[18]TAN J,ZHANG F,KARCHER D,et al.Engineering of high-precision base editors for site-specific single nucleotide replacement[J].Nat Commun,2019,10(1):439.
[19]CONG L,RAN F A,COX D,et al.Multiplex genome engineering using CRISPR/Cas systems[J].Science,2013,339(6121):819-23.
Claims (20)
- 一种双分子脱氨酶互补的碱基编辑系统，其特征在于，所述碱基编辑系统包括以下1)至5)中至少一项：1)碱基编辑融合蛋白A、碱基编辑融合蛋白B和向导RNA；2)包含编码碱基编辑融合蛋白A和碱基编辑融合蛋白B的核苷酸序列的表达构建体，和向导RNA；3)碱基编辑融合蛋白A、碱基编辑融合蛋白B和包含编码向导RNA的核苷酸序列的表达构建体；4)包含编码碱基编辑融合蛋白A和碱基编辑融合蛋白B的核苷酸序列的表达构建体，和包含编码向导RNA的核苷酸序列的表达构建体；5)包含编码碱基编辑融合蛋白A和碱基编辑融合蛋白B的核苷酸序列和编码向导RNA的核苷酸序列的表达构建体；其中所述的碱基编辑融合蛋白A自N端到C端依次包括第一nCas9多肽片段、柔性连接肽和第一核碱基脱氨酶多肽片段；所述碱基编辑融合蛋白B自N端到C端依次包括第二核碱基脱氨酶多肽片段、柔性连接肽和第二nCas9多肽片段；所述第一核碱基脱氨酶多肽片段和第二核碱基脱氨酶多肽片段选自相同的核碱基脱氨酶。
- 根据权利要求1所述的碱基编辑系统，其特征在于，所述第一nCas9多肽片段的氨基酸序列如SEQ ID NO:1所示；所述第二nCas9多肽片段的氨基酸序列如SEQ ID NO:2所示。
- 根据权利要求1所述的碱基编辑系统，其特征在于，所述柔性连接肽包括氨基酸序列如SEQ ID NO:3所示的32aa连接肽。
- 根据权利要求1所述的碱基编辑系统，其特征在于，所述碱基编辑融合蛋白A和碱基编辑融合蛋白B的N端或C端融合有至少一个核定位序列NLS。
- 根据权利要求1所述的碱基编辑系统，其特征在于，所述碱基编辑系统为胞嘧啶碱基编辑器系统；所述核碱基脱氨酶为胞苷脱氨酶。
- 根据权利要求5所述的碱基编辑系统，其特征在于，所述胞苷脱氨酶选 自：由APOBEC3A(A3A)、APOBEC3B(A3B)、APOBEC3C(A3C)、APOBEC3D(A3D)、APOBEC3F(A3F)、APOBEC3G(A3G)、APOBEC3H(A3H)、APOBEC1(A1)、APOBEC3(A3)、APOBEC2(A2)、APOBEC4(A4)和AICDA(AID)或通过单个或多个氨基酸组合突变APOBEC3A(A3A)、APOBEC3B(A3B)、APOBEC3C(A3C)、APOBEC3D(A3D)、APOBEC3F(A3F)、APOBEC3G(A3G)、APOBEC3H(A3H)、APOBEC1(A1)、APOBEC3(A3)、APOBEC2(A2)、APOBEC4(A4)和AICDA(AID)获得的胞苷脱氨酶变体构成的组。
- 根据权利要求5所述的碱基编辑系统，其特征在于，所述胞苷脱氨酶优选人源AID高活性变体AID10、大鼠来源的rA1、人源的hA3A、人源的hA3B。
- 根据权利要求5所述的碱基编辑系统，其特征在于，所述第一核碱基脱氨酶多肽片段包括如SEQ ID NO:4～8所示任一氨基酸序列；所述第二核碱基脱氨酶多肽片段包括如SEQ ID NO:9～13所示任一氨基酸序列。
- 根据权利要求5所述的碱基编辑系统，其特征在于，所述碱基编辑融合蛋白B的C端融合有至少一个尿嘧啶糖苷酶抑制子UGI,所述尿嘧啶糖苷酶抑制子UGI的氨基酸序列如SEQ ID NO:14所示。
- 根据权利要求5-9任一项所述的碱基编辑系统，其特征在于，所述胞嘧啶碱基编辑系统包括以下1)至5)任一项所述碱基编辑融合蛋白A和碱基编辑融合蛋白B的组合：1)氨基酸序列如SEQ ID NO:15所示的碱基编辑融合蛋白A：Split-AID10-N，和氨基酸序列如SEQ ID NO:16所示的碱基编辑融合蛋白B：Split-AID10-C；2)氨基酸序列如SEQ ID NO:17所示的碱基编辑融合蛋白A：Split-AID10-N5，和氨基酸序列如SEQ ID NO:18所示的碱基编辑融合蛋白B：Split-AID10-C4；3)氨基酸序列如SEQ ID NO:19所示的碱基编辑融合蛋白A：Split-BE3-N，和氨基酸序列如SEQ ID NO:20所示的碱基编辑融合蛋白B：Split-BE3-C；4)氨基酸序列如SEQ ID NO:21所示的碱基编辑融合蛋白A：Split-A3A-N，和氨基酸序列如SEQ ID NO:22所示的碱基编辑融合蛋白B：Split-A3A-C；5)氨基酸序列如SEQ ID NO:23所示的碱基编辑融合蛋白A：Split-A3B-N，和氨基酸序列如SEQ ID NO:24所示的碱基编辑融合蛋白B：Split-A3B-C。
- 根据权利要求1所述的碱基编辑系统，其特征在于，所述碱基编辑系统为腺嘌呤碱基编辑器系统；所述核碱基脱氨酶为腺苷脱氨酶。
- 根据权利要求11所述的碱基编辑系统，其特征在于，所述腺苷脱氨酶为DNA依赖型腺苷脱氨酶；优选地，所述腺苷脱氨酶为单链DNA依赖型腺苷脱氨酶。
- 根据权利要求11所述的碱基编辑系统，其特征在于，所述腺苷脱氨酶包括大肠杆菌tRNA腺苷脱氨酶TadA的变体；优选地，所述变体包括TadA-7.10、TadA-8s或TadA-8e；更优选地，所述变体为TadA-8e。
- 根据权利要求11所述的碱基编辑系统，其特征在于，所述第一核碱基多肽片段包括如SEQ ID NO:25～26所示任一氨基酸序列；所述第二核碱基多肽片段包括如SEQ ID NO:27～28所示任一氨基酸序列。
- 根据权利要求11-14任一项所述的碱基编辑系统，其特征在于，所述腺嘌呤碱基编辑系统包括以下1)至2)任一项所述碱基编辑融合蛋白A和碱基编辑融合蛋白B的组合：1)氨基酸序列如SEQ ID NO:29所示的碱基编辑融合蛋白A：Split-ABE8e-N，和氨基酸序列如SEQ ID NO:30所示的碱基编辑融合蛋白B：Split-ABE8e-C；2)氨基酸序列如SEQ ID NO:31所示的碱基编辑融合蛋白A：Split-ABE8e-N7，和氨基酸序列如SEQ ID NO:32所示的碱基编辑融合蛋白B：Split-ABE8e-C2。
- 根据权利要求1所述的碱基编辑系统，其特征在于，所述编码碱基编辑融合蛋白A和碱基编辑融合蛋白B的核苷酸序列和/或所述编码向导RNA的核苷酸序列与表达调控元件可操作地连接。
- 根据权利要求16所述的碱基编辑系统，其特征在于，所述调控元件为 启动子；所述启动子包括病毒35S启动子，玉米Ubi-1启动子，水稻Ubi启动子，病毒CMV启动子，酵母TDH3启动子，酵母GAL1启动子，拟南芥卵细胞特异的EC1.2en+EC1.1嵌合启动子，水稻U6启动子，拟南芥U6启动子酵母U6启动子或人U6启动子。
- 根据权利要求1～17任一项所述的碱基编辑系统在产生经遗传修饰的生物体中的应用，其特征在于，将所述碱基编辑系统导入生物体细胞，由所述向导RNA将所述碱基编辑器靶向细胞基因组中的靶序列，导致所述靶序列中至少一个C碱基被T碱基取代或至少一个A碱基被G碱基取代。
- 根据权利要求18所述的应用，其特征在于，所述生物为真核生物；所述真核生物为真菌、动物或植物。
- 根据权利要求19所述的应用，其特征在于，所述真菌包括酵母；所述动物包括人、小鼠、大鼠、猴、犬、猪、羊、牛或猫；所述植物包括拟南芥、水稻、小麦、玉米、大豆、向日葵、高粱、油菜、苜蓿、棉花、大麦、粟、甘蔗、番茄、烟草、木薯或马铃薯。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210503831.5 | 2022-05-10 | ||
| CN202210503831.5A CN114686456B (zh) | 2022-05-10 | 2022-05-10 | 基于双分子脱氨酶互补的碱基编辑系统及其应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2023216415A1 true WO2023216415A1 (zh) | 2023-11-16 |
Family
ID=82145309
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2022/106402 WO2023216415A1 (zh) | 2022-05-10 | 2022-07-19 | 基于双分子脱氨酶互补的碱基编辑系统及其应用 |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN114686456B (zh) |
| WO (1) | WO2023216415A1 (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114686456B (zh) * | 2022-05-10 | 2023-02-17 | 中山大学 | 基于双分子脱氨酶互补的碱基编辑系统及其应用 |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110835629A (zh) * | 2018-08-15 | 2020-02-25 | 华东师范大学 | 一种新型碱基转换编辑系统的构建方法及其应用 |
| CN110835632A (zh) * | 2018-08-15 | 2020-02-25 | 华东师范大学 | 新型碱基转换编辑系统用于基因治疗的应用 |
| CN110997728A (zh) * | 2017-05-25 | 2020-04-10 | 通用医疗公司 | 二分型碱基编辑器(bbe)结构和ii-型-cas9锌指编辑 |
| CN112266420A (zh) * | 2020-10-30 | 2021-01-26 | 华南农业大学 | 一种植物高效胞嘧啶单碱基编辑器及其构建与应用 |
| WO2021158921A2 (en) * | 2020-02-05 | 2021-08-12 | The Broad Institute, Inc. | Adenine base editors and uses thereof |
| CN114072509A (zh) * | 2019-01-31 | 2022-02-18 | 比姆医疗股份有限公司 | 脱氨反应脱靶减低的核碱基编辑器和使用其修饰核碱基靶序列的方法 |
| CN114686456A (zh) * | 2022-05-10 | 2022-07-01 | 中山大学 | 基于双分子脱氨酶互补的碱基编辑系统及其应用 |
Family Cites Families (30)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20180096800A (ko) * | 2016-01-11 | 2018-08-29 | 더 보드 어브 트러스티스 어브 더 리랜드 스탠포드 주니어 유니버시티 | 키메라 단백질 및 유전자 발현을 조절하는 방법 |
| AU2017358264A1 (en) * | 2016-11-14 | 2019-02-21 | Institute Of Genetics And Developmental Biology, Chinese Academy Of Sciences | A method for base editing in plants |
| WO2019023680A1 (en) * | 2017-07-28 | 2019-01-31 | President And Fellows Of Harvard College | METHODS AND COMPOSITIONS FOR EVOLUTION OF BASIC EDITORS USING PHAGE-ASSISTED CONTINUOUS EVOLUTION (PACE) |
| CN111373041A (zh) * | 2017-09-26 | 2020-07-03 | 伊利诺伊大学理事会 | 用于基因组编辑和调节转录的crispr/cas系统和方法 |
| CN107722125B (zh) * | 2017-09-28 | 2021-05-07 | 中山大学 | 一种人工转录激活因子dCas9-TV及其编码基因与应用 |
| CN110157727A (zh) * | 2017-12-21 | 2019-08-23 | 中国科学院遗传与发育生物学研究所 | 植物碱基编辑方法 |
| AR114014A1 (es) * | 2017-12-22 | 2020-07-08 | Inst Genetics & Developmental Biology Cas | Sistema de edición de bases y método basado en proteína cpf1 |
| CN110835634B (zh) * | 2018-08-15 | 2022-07-26 | 华东师范大学 | 一种新型碱基转换编辑系统及其应用 |
| WO2020049158A1 (en) * | 2018-09-07 | 2020-03-12 | Astrazeneca Ab | Compositions and methods for improved nucleases |
| AU2020223306A1 (en) * | 2019-02-13 | 2021-08-05 | Beam Therapeutics Inc. | Methods of editing a disease-associated gene using adenosine deaminase base editors, including for the treatment of genetic disease |
| CN114190093A (zh) * | 2019-02-13 | 2022-03-15 | 比姆医疗股份有限公司 | 使用腺苷酸脱氨酶碱基编辑器破坏疾病相关基因的剪接受体位点，包括用于治疗遗传性疾病 |
| JP2022520081A (ja) * | 2019-02-13 | 2022-03-28 | ビーム セラピューティクス インク． | アデノシンデアミナーゼ塩基エディターおよびそれを用いて標的配列中の核酸塩基を改変する方法 |
| CN111718420B (zh) * | 2019-03-19 | 2021-09-14 | 华东师范大学 | 一种用于基因治疗的融合蛋白及其应用 |
| EP3956349A1 (en) * | 2019-04-17 | 2022-02-23 | The Broad Institute, Inc. | Adenine base editors with reduced off-target effects |
| CN112239756B (zh) * | 2019-07-01 | 2022-04-19 | 科稷达隆(北京)生物技术有限公司 | 一组来源于植物的胞嘧啶脱氨酶和其在碱基编辑系统中的应用 |
| CN112266418A (zh) * | 2019-07-08 | 2021-01-26 | 中国科学院微生物研究所 | 改进的基因组编辑系统及其应用 |
| WO2021042062A2 (en) * | 2019-08-30 | 2021-03-04 | Joung J Keith | Combinatorial adenine and cytosine dna base editors |
| EP4022053A4 (en) * | 2019-08-30 | 2023-05-31 | The General Hospital Corporation | DNA BASE EDITORS WITH C-TO-G TRANSVERSION |
| CN112979821B (zh) * | 2019-12-18 | 2022-02-08 | 华东师范大学 | 一种提高基因编辑效率的融合蛋白及其应用 |
| EP4093879A4 (en) * | 2020-01-25 | 2024-02-28 | Univ Pennsylvania | COMPOSITIONS FOR SMALL MOLECULE CONTROL OF PRECISE BASE EDITING OF TARGET NUCLEIC ACIDS AND METHODS OF USE THEREOF |
| BR112022017704A2 (pt) * | 2020-03-04 | 2022-11-01 | Suzhou Qi Biodesign Biotechnology Company Ltd | Método e sistema de edição de genoma multiplex |
| EP4130257A4 (en) * | 2020-03-04 | 2024-05-01 | Suzhou Qi Biodesign Biotechnology Company Ltd | IMPROVED CYTOSINE BASE EDITING SYSTEM |
| CN114058604B (zh) * | 2020-03-10 | 2023-05-05 | 上海科技大学 | 一种融合蛋白及其在碱基编辑中的用途 |
| CN111748578B (zh) * | 2020-07-14 | 2023-08-25 | 北大荒垦丰种业股份有限公司 | 植物导引模板原位合成基因编辑方法及应用 |
| KR20230084505A (ko) * | 2020-09-11 | 2023-06-13 | 라이프에디트 테라퓨틱스, 인크. | Dna 변형 효소 및 그의 활성 단편 및 변이체 및 사용 방법 |
| KR20230068402A (ko) * | 2020-09-18 | 2023-05-17 | 기초과학연구원 | 표적화된 탈아미노효소 및 이를 이용한 염기 교정 |
| CN112746083B (zh) * | 2020-12-11 | 2023-08-11 | 中山大学 | 一种通过单碱基编辑靶基因启动子失活基因的方法 |
| CN113667017A (zh) * | 2021-07-28 | 2021-11-19 | 上海南方模式生物科技股份有限公司 | 一种可提高CRISPR/Cas9系统同源重组效率的方法和应用 |
| CN114480391B (zh) * | 2022-01-26 | 2024-03-29 | 中国科学技术大学 | 用于提高CRISPR/Cas9系统基因编辑效率的启动子及其应用 |
| CN114835821B (zh) * | 2022-04-18 | 2023-12-22 | 上海贝斯昂科生物科技有限公司 | 一种高效特异实现碱基颠换的编辑系统、方法及用途 |
-
2022
- 2022-05-10 CN CN202210503831.5A patent/CN114686456B/zh active Active
- 2022-07-19 WO PCT/CN2022/106402 patent/WO2023216415A1/zh unknown
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110997728A (zh) * | 2017-05-25 | 2020-04-10 | 通用医疗公司 | 二分型碱基编辑器(bbe)结构和ii-型-cas9锌指编辑 |
| CN110835629A (zh) * | 2018-08-15 | 2020-02-25 | 华东师范大学 | 一种新型碱基转换编辑系统的构建方法及其应用 |
| CN110835632A (zh) * | 2018-08-15 | 2020-02-25 | 华东师范大学 | 新型碱基转换编辑系统用于基因治疗的应用 |
| CN114072509A (zh) * | 2019-01-31 | 2022-02-18 | 比姆医疗股份有限公司 | 脱氨反应脱靶减低的核碱基编辑器和使用其修饰核碱基靶序列的方法 |
| WO2021158921A2 (en) * | 2020-02-05 | 2021-08-12 | The Broad Institute, Inc. | Adenine base editors and uses thereof |
| CN112266420A (zh) * | 2020-10-30 | 2021-01-26 | 华南农业大学 | 一种植物高效胞嘧啶单碱基编辑器及其构建与应用 |
| CN114686456A (zh) * | 2022-05-10 | 2022-07-01 | 中山大学 | 基于双分子脱氨酶互补的碱基编辑系统及其应用 |
Non-Patent Citations (2)
| Title |
|---|
| KANTOR ARIEL, MCCLEMENTS MICHELLE, MACLAREN ROBERT: "CRISPR-Cas9 DNA Base-Editing and Prime-Editing", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, MOLECULAR DIVERSITY PRESERVATION INTERNATIONAL (MDPI), BASEL, CH, vol. 21, no. 17, 1 September 2020 (2020-09-01), Basel, CH , pages 6240, XP055902933, ISSN: 1661-6596, DOI: 10.3390/ijms21176240 * |
| XU XIN, LIU MINGJUN: "Recent advances and applications of base editing systems", SHENG WU GONG CHENG XUE BAO = CHINESE JOURNAL OF BIOTECHNOLOGY, vol. 37, no. 7, 25 July 2021 (2021-07-25), pages 2307 - 2321, XP093106749, DOI: 10.13345/j.cjb.200480 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114686456A (zh) | 2022-07-01 |
| CN114686456B (zh) | 2023-02-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108795972B (zh) | 不使用转基因标记序列分离细胞的方法 | |
| AU2013256240B2 (en) | Targeted modification of malate dehydrogenase | |
| JP2015500648A (ja) | 所定の標的核酸配列を修飾するための組成物及び方法 | |
| JP2022511508A (ja) | ゲノム編集による遺伝子サイレンシング | |
| US20210087557A1 (en) | Methods and compositions for targeted genomic insertion | |
| WO2023216415A1 (zh) | 基于双分子脱氨酶互补的碱基编辑系统及其应用 | |
| KR20220137166A (ko) | 전이유전자성 마커 서열을 이용하지 않는 세포 단리 방법 | |
| US11913004B2 (en) | Plant promoter for transgene expression | |
| US20220098606A1 (en) | Plant promoter for transgene expression | |
| JP2022549430A (ja) | Ｄｎａ塩基編集のための方法及び組成物 | |
| JP2022522823A (ja) | 天然ｍｉＲＮＡのゲノム編集による標的遺伝子発現の抑制 | |
| CN108473997B (zh) | 用于转基因表达的植物启动子 | |
| US20200392524A1 (en) | Plant promoter for transgene expression | |
| US20220090111A1 (en) | Plant promoter for transgene expression | |
| US11459577B2 (en) | Targeted insertion sites in the maize genome | |
| CN110129359B (zh) | 检测基因编辑事件以及测定基因编辑效率的方法以及其应用 | |
| US20120070900A1 (en) | T-dna/protein nano-complexes for plant transformation | |
| WO2023230459A2 (en) | Compositions and methods for targeting donor polynucelotides in soybean genomic loci | |
| US20230114951A1 (en) | Targeted insertion sites in the maize genome |
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: 22941359 Country of ref document: EP Kind code of ref document: A1 |