CRISPR/Cas9技术存在的问题及其改进措施的研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2017.04.009

摘要/Abstract

摘要： CRISPR/Cas9技术是一种RNA引导的基因组编辑技术,能对基因进行精确性敲除、敲入、替换等,从而实现探究基因功能、修复致病基因等目的。该技术凭借操作简便、价格低廉、可对基因位点进行编辑、可拓展性强等优势,在近几年迅速发展完善,已成为继锌指核酸酶技术和类转录激活因子效应物核酸酶技术之后的第三大基因组编辑技术。在简单介绍CRISPR/Cas9技术的作用机制后,主要针对该技术现阶段面临的精确修复比例低、PAM限制识别序列、脱靶现象、马赛克现象的问题以及相应的改进措施进行阐述,旨在让研究者客观的认识CRISPR/Cas9技术存在的不足之处,为合理使用或改进该技术提供系统的文献综述。

关键词: CRISPR/Cas9, 基因编辑, 精确编辑, 脱靶, 马赛克现象

Abstract: CRISPR/Cas9[clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9]is a RNA-guided genome-editing technique,which can be applied in the deletion,insertion or replacement of specific DNA sequence thus to verify the function of genes or remedy pathogenic genes. Owing to its simplicity,affordability,multiple-site editing and scalability,the technique of CRISPR/Cas9 is undergoing rapid development and perfection and has been one of the first three prevailing genome-editing technology following that of Zinc finger nucleases and transcription activator-like effector nucleases. In this review,we focus on the current issues of CRISPR/Cas9,including low efficiency of precise-editing,sequence-recognizing limitation from PAM,off-target,unavoidable mosaicism,together with corresponding tactics for improvement. Our aim is to provide the objective understanding on the current disadvantage of CRISPR/Cas9 technique to researchers and the systematic review for reasonable application and improvement of this technology.

Key words: CRISPR/Cas9, genome-editing, precise-editing, off-target, mosaicism

袁伟曦, 喻云梅, 胡春财, 赵祖国. CRISPR/Cas9技术存在的问题及其改进措施的研究进展[J]. 生物技术通报, 2017, 33(4): 70-77.

YUAN Wei-xi, YU Yun-mei, HU Chun-cai, ZHAO Zu-guo. Current Issues and Progress in the Application of CRISPR/Cas9 Technique[J]. Biotechnology Bulletin, 2017, 33(4): 70-77.

参考文献

[1] Gao SL, Tong YY, Wen ZQ, et al. Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system[J]. J Ind Microbiol Biotechnol. 2016, 43（8）：1085-1093.
[2] Cheng M, Jin XB, Mu L, et al. Combination of the clustered regularly interspaced short palindromic repeats（CRISPR）-associated 9 technique with the piggybac transposon system for mouse in utero electroporation to study cortical development[J]. J Neurosci Res, 2016, 94（9）：814-824.
[3] Bi YZ, Hua ZD, Liu XM, et al. Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP[J]. Sci Rep, 2016, 6：31729.
[4] Jiang WY, Bikard D, Cox D, et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems[J]. Nature Biotechnology, 2013, 31（3）：233-239.
[5] Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J]. Science, 2012, 337（6096）：816-821.
[6] Jinek M, Jiang F, Taylor DW, et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation[J]. Science, 2014, 343（6176）：1247997.
[7] Nishimasu H, Ran FA, Hsu PD, et al. Crystal structure of Cas9 in complex with guide RNA and target DNA[J]. Cell, 2014, 156（5）：935-949.
[8] Sternberg SH, Redding S, Jinek M, et al. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9[J]. Nature, 2014, 507（7490）：62-67.
[9] Anders C, Niewoehner O, Duerst A, et al. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease[J]. Nature, 2014, 513（7519）：569-573.
[10] Jiang FG, Taylor DW, Chen JS, et al. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage[J]. Science, 2016, 351（6275）：867-871.
[11] Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering[J]. Cell, 2014, 6：1262-1278.
[12] Gratz SJ, Ukken FP, Rubinstein CD, et al. Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila[J]. Genetics, 2014, 196（4）：961-971.
[13] Miyaoka Y, Berman JR, Cooper SB, et al. Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing[J]. Sci Rep, 2016, 6：23549.
[14] Paquet D, Kwart D, Chen A, et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9[J]. Nature, 2016, 533（7601）：125-129.
[15] Ma YW, Chen W, Zhang X, et al. Increasing the efficiency of CRISPR/Cas9-mediated precise genome editing in rats by inhibiting NHEJ and using Cas9 protein[J]. RNA Biology, 2016, 13（7）：605-612.
[16] Lin CL, Li HH, Hao MR, et al. Increasing the efficiency of CRISPR/Cas9-mediated precise genome editing of HSV-1 virus in human cells[J]. Sci Rep, 2016, 6：34531.
[17] Chu VT, Weber T, Wefers B, et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-inducedprecise gene editing in mammalian cells[J]. Nat Biotechnol, 2015, 33（5）：543-548.
[18] 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.
[19] Yang D, Scavuzzo MA, Chmielowiec J, et al. Enrichment of G2/M cell cyclephase in human pluripotent stemcells enhances HDR-mediated gene repair with customizable endonucleases[J]. Scientific Reports, 2016, 6：21264.
[20] 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]. Nature Biotechnology, 2016, 34（3）：339-344.
[21] Hendel A, Bak RO, Clark JT, et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells[J]. Nature Biotechnology, 2015, 33（9）：985-989.
[22] Yu C, Liu YX, Ma TH, et al. Small molecules enhance CRISPR genome editing in pluripotent stem cells[J]. Cell Stem Cell, 2015, 16（2）：142-147.
[23] Nishida K, Arazoe T, Yachie N, et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems[J]. Science, 2016, 353（6305）：1248.
[24] Komor AC, Kim YB, Packer MS, et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage[J]. Nature, 2016, 533（7603）：420-424.
[25] Xie KB, et al. Genome-wide prediction of highly specific guide RNA spacers for CRISPR-Cas9-mediated genome editing in model plants and major crops[J]. Mol Plant, 2014, 5：923-926.
[26] Anders C, Bargsten K, Jinek M, et al. Structural plasticity of PAM recognition by engineered variants of the RNA-guided endonuclease Cas9[J]. Mol Cell, 2016, 61（6）：895-902.
[27] Kleinstiver BP, Prew MS, Tsai SQ, et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities[J]. Nature, 2015, 523（7561）：481-485.
[28] Chylinski K, Makarova KS, Charpentier E, et al. Classification and evolutionof type II CRISPR-Cas systems[J]. Nucleic Acids Res, 2014, 42（10）：6091-6105.
[29] Hirano H, Gootenberg JS, Horii T, et al. Structure and Engineering of Francisella novicida Cas9[J]. Cell, 2016, 5：950-961.
[30] Feng Y, Chen C, Han YX, et al. Expanding CRISPR/Cas9 genome editing capacity in zebrafish using saCas9[J]. G3（Bethesda）, 2016, 6（8）：2517-2521.
[31] Lee CM, Cradick TJ, Bao G, et al. The Neisseria meningitidis CR-ISPR-Cas9 system enables specific genome editing in mammalian cells[J]. Mol Ther, 2016, 24（3）：645-54.
[32] Li W, Bian X, et al. Comparative analysis of clustered regularly interspaced short palindromic repeats （CRISPR）of Streptococcus thermophilus St-I and its bacteriophage-insensitive mutants（BIM）derivatives[J]. Curr Microbiol, 2016, 73（3）：393-400.
[33] Glemzaite M, Balciunaite E, Karvelis T, et al. Targeted gene editing by transfection of in vitro reconstituted Streptococcus thermophilus Cas9 nuclease complex[J]. RNA Biol, 2015, 12（1）：1-4.
[34] Fonfara I, Le Rhun A, Chylinski K, et al. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems[J]. Nucleic Acids Res, 2014, 42（4）：2577-2590.
[35] Kleinstiver BP, Prew MS, Tsai SQ, et al. Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition[J]. Nat Biotechnol, 2015, 33（12）：1293-1298.
[36] Hirano H, Gootenberg JS, Horii T, et al. Structure and engineering of Francisella novicida Cas9[J]. Cell, 2016, 5：950-961.
[37] Abudayyeh OO, Gootenberg JS, Konermann S, et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector[J]. Science, 2016, 353（6299）：557.
[38] Chiang TW, Ie Sage C, Larrieu D, et al. CRISPR-Cas9（D10A）nickase-based genotypic and phenotypic screening to enhance genome editing[J]. Sci Rep, 2016, 6：24356.
[39] Zhang B, Liu ZQ, Liu C, et al. Application of CRISPRi in Corynebacterium glutamicum for shikimic acid production[J]. Biotechnol Lett, 2016, 38（12）：2153-2161.
[40] Pan Y, Shen N, Jung-Klawitter S, et al. CRISPR RNA-guided FokI nucleases repair a PAH variant in a phenylketonuria model[J]. Sci Rep, 2016, 6：35794.
[41] Terao M, Tamano M, Hara S, et al. Utilization of the CRISPR/Cas9 system for the efficient production of mutant mice using crRNA/tracrRNA with Cas9 nickase and FokI-dCas9[J]. Exp Anim, 2016, 65（3）：275-283.
[42] Slaymaker IM, et al. Rationally engineered Cas9 nucleases with improved specificity[J]. Science, 2016, 351（6288）：84-88.
[43] Kleinstiver BP, Pattanayak V, Prew MS, et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects[J]. Nature, 2016, 529（7587）：490-495.
[44] Cao J, Wu LZ, Zhang SM, et al. An easy and efficient inducible CRISPR/Cas9 platform with improved specificity for multiple gene targeting[J]. Nucleic Acids Res, 2016, 44（19）：e149.
[45] Davis KM, Pattanayak V, Thompson DB, et al, Small molecule-triggered Cas9 protein with improved genome-editing specificity[J]. Nat Chem Biol, 2015, 11（5）：316-318.
[46] Fu YF, Sander JD, Reyon D, et al. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs[J]. Nature Biotechnology, 2014, 32（3）：279-284.
[47] Cho SW, Kim S, Kim Y, et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases[J]. Genome Res, 2014, 24（1）：132-141.
[48] Lee CM, Cradick TJ, Bao G. The Neisseria meningitidis CRISPR-Cas9 system enables specific genome editing in mammalian cells[J]. Mol Ther, 2016, 24（3）：645-654.
[49] Müller M, Lee CM, Gasiunas G, et al. Streptococcus thermophilus CRISPR-Cas9 systems enable specific editing of the human genome [J]. Mol Ther, 2016, 24（3）：636-644.
[50] Kalebic N, Taverna E, Tavano S, et al. CRISPR/Cas9-induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo[J]. EMBO Rep, 2016, 3：338-348.
[51] Hara S, et al. Microinjection-based generation of mutant mice with a double mutation and a 0. 5 Mb deletion in their genome by the CR-ISPR/Cas9 system[J]. J Reprod Dev, 2016, 62（5）：531-536.
[52] 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, 6：1012-1019.
[53] Liang PP, Xu YW, Zhang XY, et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes[J]. Protein Cell, 2015, 6（5）：363-372.
[54] Hashimoto M, Yamashita Y, Takemoto T. Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse[J]. Dev Biol, 2016, 418（1）：1-9.
[55] Yang LH, Güell M, Niu D, et al. Genome-wide inactivation of porcine endogenous retroviruses（PERVs）[J]. Science, 2015, 350（6264）：1101-1104.
[56] Bialek JK, Dunay GA, Voges M, et al. Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems[J]. PLoS One, 2016, 11（6）：e0158294.
[57] Zhu W, Xie K, Xu YJ, et al. CRISPR/Cas9 produces anti-hepatitis B virus effect in hepatoma cells and transgenic mouse[J]. Virus Res, 2016, 217：125-132.
[58] Khatodia S, Bhatotia K, Passricha N, et al. The CRISPR/Cas genome-editing tool：application in improvement of crops[J]. Front Plant Sci, 2016, 7：506.
[59] Bassuk AG, Zheng A, Li Y, et al. Precision Medicine：Genetic Repair of Retinitis Pigmentosa in Patient-Derived Stem Cells[J]. Sci Rep, 2016, 6：19969.