基于CRISPR/Cas9技术的基因敲入/敲除策略
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摘要
成簇的规律间隔性短回文序列(CRISPR)基因编辑系统,因其设计简单操作方便和无种属限制,已成为一种广泛应用的基因组定点编辑工具,在复杂的基因组编辑,例如基因的人源化改造以及条件等位基因的构建中有所应用。在自然界中,CRISPR系统拥有多种类别。其中,CRISPR/Cas9系统是研究最深入、应用最成熟的一种。本文针对CRISPR/Cas9系统,分别从基因敲入/敲除片段的大小、同源臂长短、构型即递送方式等技术环节进行综述,阐述不同设计及操作条件下由CRISPR/Cas9系统介导的基因敲入/敲除的效率差异。
CRISPR(cluster regularly interspaced short palindrome repeats) gene editing system has become a widely used genome editing tool because of its remarkable advantages such as relatively easy in design and production, as well as no species limit. It has been used in complex genome editing, such as humanization of genes and the construction of conditional alleles. CRISPR system has a variety of categories in nature. CRISPR/Cas9 system is one of the most in-depth studies and the most mature technologies. In this review, we mainly focus on the important structure features of CRISPR/Cas9, such as gene editing fragment, length of homologous arms and its corresponding structure, as well as its delivery methods. The difference in efficiency of gene knockin/knockout mediated by the CRISPR/Cas9 system under different design and operating conditions was also summarized.
CRISPR(cluster regularly interspaced short palindrome repeats) gene editing system has become a widely used genome editing tool because of its remarkable advantages such as relatively easy in design and production, as well as no species limit. It has been used in complex genome editing, such as humanization of genes and the construction of conditional alleles. CRISPR system has a variety of categories in nature. CRISPR/Cas9 system is one of the most in-depth studies and the most mature technologies. In this review, we mainly focus on the important structure features of CRISPR/Cas9, such as gene editing fragment, length of homologous arms and its corresponding structure, as well as its delivery methods. The difference in efficiency of gene knockin/knockout mediated by the CRISPR/Cas9 system under different design and operating conditions was also summarized.
引文
[1] Wen K, Yang L, Xiong T, et al. Critical roles of long noncoding RNAs in Drosophila spermatogenesis [J]. Genome Res, 2016, 26(9): 1233-1244
[2] Liu P, Long L, Xiong K, et al. Heritable/conditional genome editing in C. elegans using a CRISPR-Cas9 feeding system [J]. Cell Res, 2014, 24(7): 886-889
[3] Yang H, Wang H, Shivalila C S, et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering [J]. Cell, 2013, 154(6): 1370-1379
[4] Zuo E, Cai Y J, Li K, et al. One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs [J]. Cell Res, 2017, 27(7): 933-945
[5] Varshney GK, Pei W, LaFave MC, et al. High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9 [J]. Genome Res, 2015, 25(7): 1030-1042
[6] Lv Q, Yuan L, Deng J, et al. Efficient Generation of Myostatin Gene Mutated Rabbit by CRISPR/Cas9 [J]. Sci Rep, 2016, 6: 25029
[7] Wang K, Ouyang H, Xie Z, et al. Efficient Generation of Myostatin Mutations in Pigs Using the CRISPR/Cas9 System [J]. Sci Rep, 2015, 5: 16623
[8] Wu Y, Liang D, Wang Y, et al. Correction of a genetic disease in mouse via use of CRISPR-Cas9 [J]. Cell Stem Cell,2013, 13(6): 659-662
[9] Fujii W, Kawasaki K, Sugiura K, et al. Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease [J]. Nucleic Acids Res, 2013, 41(20): e187
[10] Zheng Q, Cai X, Tan MH, et al. Precise gene deletion and replacement using the CRISPR/Cas9 system in human cells [J]. Biotechniques, 2014, 57(3): 115-124
[11] Chen X, Xu F, Zhu C, et al. Dual sgRNA-directed gene knockout using CRISPR/Cas9 technology in Caenorhabditis elegans [J]. Sci Rep, 2014, 4: 7581
[12] Song Y, Yuan L, Wang Y, et al. Efficient dual sgRNA-directed large gene deletion in rabbit with CRISPR/Cas9 system [J]. Cell Mol Life Sci, 2016, 73(15): 2959-2968
[13] Kim S, Kim D, Cho SW, et al. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins [J]. Genome Res, 2014, 24(6): 1012-1019
[14] Wang L, Shao Y, Guan Y, et al. Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos [J]. Sci Rep, 2015, 5: 17517
[15] DeWitt MA, Magis W, Bray NL, et al. Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells [J]. Sci Transl Med, 2016, 8(360): 360ra134
[16] Lin Z, Hsu PJ, Xing X, et al. Mettl3-/Mettl14-mediated mRNA N6-methyladenosine modulates murine spermatogenesis [J]. Cell Res, 2017, 27(10): 1216-1230
[17] Ma Y, Ma J, Zhang X, et al. Generation of eGFP and Cre knockin rats by CRISPR/Cas9 [J]. FEBS J, 2014, 281(17): 3779-3790
[18] Zhang JP, Li XL, Li GH, et al. Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9- mediated double-stranded DNA cleavage [J]. Genome Biol, 2017, 18(1): 35
[19] Cristea S, Freyvert Y, Santiago Y, et al. In vivo cleavage of transgene donors promotes nuclease-mediated targeted integration [J]. Biotechnol Bioeng, 2013, 110(3): 871-880
[20] Yoshimi K, Kunihiro Y, Kaneko T, et al. ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes [J]. Nat Commun, 2016, 7: 10431
[21] Yang L, Guell M, Byrne S, et al. Optimization of scarless human stem cell genome editing [J]. Nucleic Acids Res, 2013, 41(19): 9049-9061
[22] Chen F, Pruett-Miller SM, Huang Y, et al. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases [J]. Nat Methods, 2011, 8(9): 753-755
[23] Radecke S, Radecke F, Cathomen T, et al. Zinc-finger nuclease-induced gene repair with oligodeoxynucleotides: wanted and unwanted target locus modifications [J]. Mol Ther, 2010, 18(4): 743-753
[24] Bottcher R, Hollmann M, Merk K, et al. Efficient chromosomal gene modification with CRISPR/cas9 and PCR- based homologous recombination donors in cultured Drosophila cells [J]. Nucleic Acids Res, 2014, 42(11): e89
[25] Yin H, Xue W, Chen S, et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype [J]. Nat Biotechno, 2014, 32(6): 551-553
[26] Richardson CD, Ray GJ, DeWitt MA, et al. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA [J]. Nat Biotechnol, 2016, 34(3): 339-344
[27] Shy BR, MacDougall MS, Clarke R, et al. Co-incident insertion enables high efficiency genome engineering in mouse embryonic stem cells [J]. Nucleic Acids Res, 2016, 44(16): 7997-8010
[28] Ishizu T, Higo S, Masumura Y, et al. Targeted Genome Replacement via Homology-directed Repair in Non-dividing Cardiomyocytes [J]. Sci Rep, 2017, 7(1): 9363
[29] Yao X, Wang X, Hu X, et al. Homology-mediated end joining-based targeted integration using CRISPR/Cas9 [J]. Cell Res, 2017, 27(6): 801-814
[30] Burger A, Lindsay H, Felker A, et al. Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes [J].Development, 2016, 143(11): 2025-2037
[31] Aida T, Chiyo K, Usami T, et al. Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice [J]. Genome Biol, 2015, 16: 87
[32] Schumann K, Lin S, Boyer E, et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins [J]. Proc Natl Acad Sci U S A, 2015, 112(33): 10437-10442
[33] Ramakrishna S, Kwaku Dad AB, Beloor J, et al. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA [J]. Genome Res, 2014, 24(6): 1020-1027
[34] Horii T, Arai Y, Yamazaki M, et al. Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering [J]. Sci Rep, 2014, 4: 4513
[35] Wang H, Yang H, Shivalila CS, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering [J]. Cell, 2013, 153(4): 910-918
[36] Mali P, Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9 [J]. Science, 2013, 339(6121): 823-826
[37] Byrne SM, Ortiz L, Mali P, et al. Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells [J]. Nucleic Acids Res, 2015, 43(3): e21
[38] Maruyama T, Dougan SK, Truttmann MC, et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining [J]. Nat Biotechnol, 2015, 33(5): 538-542
[39] Pinder J, Salsman J, Dellaire G. Nuclear domain ‘knock-in’ screen for the evaluation and identification of small molecule enhancers of CRISPR-based genome editing [J]. Nucleic Acids Res, 2015, 43(19): 9379-9392
[40] Robert F, Barbeau M, Ethier S, et al. Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing [J]. Genome Med, 2015, 7: 93
[41] Song J, Yang D, Xu J, et al. RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency [J]. Nat Commun, 2016, 7: 10548
[42] Lin S, Staahl BT, Alla RK, et al. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery [J]. Elife, 2014, 3: e04766
[43] Fu Y, Foden JA, Khayter C, et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells [J]. Nat Biotechnol, 2013, 31(9): 822-826
[44] Hu JH, Miller SM, Geurts MH, et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity [J]. Nature, 2018, 556(7699): 57-63
[2] Liu P, Long L, Xiong K, et al. Heritable/conditional genome editing in C. elegans using a CRISPR-Cas9 feeding system [J]. Cell Res, 2014, 24(7): 886-889
[3] Yang H, Wang H, Shivalila C S, et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering [J]. Cell, 2013, 154(6): 1370-1379
[4] Zuo E, Cai Y J, Li K, et al. One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs [J]. Cell Res, 2017, 27(7): 933-945
[5] Varshney GK, Pei W, LaFave MC, et al. High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9 [J]. Genome Res, 2015, 25(7): 1030-1042
[6] Lv Q, Yuan L, Deng J, et al. Efficient Generation of Myostatin Gene Mutated Rabbit by CRISPR/Cas9 [J]. Sci Rep, 2016, 6: 25029
[7] Wang K, Ouyang H, Xie Z, et al. Efficient Generation of Myostatin Mutations in Pigs Using the CRISPR/Cas9 System [J]. Sci Rep, 2015, 5: 16623
[8] Wu Y, Liang D, Wang Y, et al. Correction of a genetic disease in mouse via use of CRISPR-Cas9 [J]. Cell Stem Cell,2013, 13(6): 659-662
[9] Fujii W, Kawasaki K, Sugiura K, et al. Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease [J]. Nucleic Acids Res, 2013, 41(20): e187
[10] Zheng Q, Cai X, Tan MH, et al. Precise gene deletion and replacement using the CRISPR/Cas9 system in human cells [J]. Biotechniques, 2014, 57(3): 115-124
[11] Chen X, Xu F, Zhu C, et al. Dual sgRNA-directed gene knockout using CRISPR/Cas9 technology in Caenorhabditis elegans [J]. Sci Rep, 2014, 4: 7581
[12] Song Y, Yuan L, Wang Y, et al. Efficient dual sgRNA-directed large gene deletion in rabbit with CRISPR/Cas9 system [J]. Cell Mol Life Sci, 2016, 73(15): 2959-2968
[13] Kim S, Kim D, Cho SW, et al. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins [J]. Genome Res, 2014, 24(6): 1012-1019
[14] Wang L, Shao Y, Guan Y, et al. Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos [J]. Sci Rep, 2015, 5: 17517
[15] DeWitt MA, Magis W, Bray NL, et al. Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells [J]. Sci Transl Med, 2016, 8(360): 360ra134
[16] Lin Z, Hsu PJ, Xing X, et al. Mettl3-/Mettl14-mediated mRNA N6-methyladenosine modulates murine spermatogenesis [J]. Cell Res, 2017, 27(10): 1216-1230
[17] Ma Y, Ma J, Zhang X, et al. Generation of eGFP and Cre knockin rats by CRISPR/Cas9 [J]. FEBS J, 2014, 281(17): 3779-3790
[18] Zhang JP, Li XL, Li GH, et al. Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9- mediated double-stranded DNA cleavage [J]. Genome Biol, 2017, 18(1): 35
[19] Cristea S, Freyvert Y, Santiago Y, et al. In vivo cleavage of transgene donors promotes nuclease-mediated targeted integration [J]. Biotechnol Bioeng, 2013, 110(3): 871-880
[20] Yoshimi K, Kunihiro Y, Kaneko T, et al. ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes [J]. Nat Commun, 2016, 7: 10431
[21] Yang L, Guell M, Byrne S, et al. Optimization of scarless human stem cell genome editing [J]. Nucleic Acids Res, 2013, 41(19): 9049-9061
[22] Chen F, Pruett-Miller SM, Huang Y, et al. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases [J]. Nat Methods, 2011, 8(9): 753-755
[23] Radecke S, Radecke F, Cathomen T, et al. Zinc-finger nuclease-induced gene repair with oligodeoxynucleotides: wanted and unwanted target locus modifications [J]. Mol Ther, 2010, 18(4): 743-753
[24] Bottcher R, Hollmann M, Merk K, et al. Efficient chromosomal gene modification with CRISPR/cas9 and PCR- based homologous recombination donors in cultured Drosophila cells [J]. Nucleic Acids Res, 2014, 42(11): e89
[25] Yin H, Xue W, Chen S, et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype [J]. Nat Biotechno, 2014, 32(6): 551-553
[26] Richardson CD, Ray GJ, DeWitt MA, et al. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA [J]. Nat Biotechnol, 2016, 34(3): 339-344
[27] Shy BR, MacDougall MS, Clarke R, et al. Co-incident insertion enables high efficiency genome engineering in mouse embryonic stem cells [J]. Nucleic Acids Res, 2016, 44(16): 7997-8010
[28] Ishizu T, Higo S, Masumura Y, et al. Targeted Genome Replacement via Homology-directed Repair in Non-dividing Cardiomyocytes [J]. Sci Rep, 2017, 7(1): 9363
[29] Yao X, Wang X, Hu X, et al. Homology-mediated end joining-based targeted integration using CRISPR/Cas9 [J]. Cell Res, 2017, 27(6): 801-814
[30] Burger A, Lindsay H, Felker A, et al. Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes [J].Development, 2016, 143(11): 2025-2037
[31] Aida T, Chiyo K, Usami T, et al. Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice [J]. Genome Biol, 2015, 16: 87
[32] Schumann K, Lin S, Boyer E, et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins [J]. Proc Natl Acad Sci U S A, 2015, 112(33): 10437-10442
[33] Ramakrishna S, Kwaku Dad AB, Beloor J, et al. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA [J]. Genome Res, 2014, 24(6): 1020-1027
[34] Horii T, Arai Y, Yamazaki M, et al. Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering [J]. Sci Rep, 2014, 4: 4513
[35] Wang H, Yang H, Shivalila CS, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering [J]. Cell, 2013, 153(4): 910-918
[36] Mali P, Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9 [J]. Science, 2013, 339(6121): 823-826
[37] Byrne SM, Ortiz L, Mali P, et al. Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells [J]. Nucleic Acids Res, 2015, 43(3): e21
[38] Maruyama T, Dougan SK, Truttmann MC, et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining [J]. Nat Biotechnol, 2015, 33(5): 538-542
[39] Pinder J, Salsman J, Dellaire G. Nuclear domain ‘knock-in’ screen for the evaluation and identification of small molecule enhancers of CRISPR-based genome editing [J]. Nucleic Acids Res, 2015, 43(19): 9379-9392
[40] Robert F, Barbeau M, Ethier S, et al. Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing [J]. Genome Med, 2015, 7: 93
[41] Song J, Yang D, Xu J, et al. RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency [J]. Nat Commun, 2016, 7: 10548
[42] Lin S, Staahl BT, Alla RK, et al. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery [J]. Elife, 2014, 3: e04766
[43] Fu Y, Foden JA, Khayter C, et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells [J]. Nat Biotechnol, 2013, 31(9): 822-826
[44] Hu JH, Miller SM, Geurts MH, et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity [J]. Nature, 2018, 556(7699): 57-63
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