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CRISPR/Cas9系统中sgRNA设计与脱靶效应评估

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  • 1. 华中农业大学,农业动物遗传育种与繁殖教育部重点实验室,武汉 430070;
    2. 中国人民解放军第161医院妇产科,武汉 430010
谢胜松,副研究员,研究方向:动物功能基因组与疾病诊治。E-mail: ssxie@mail.hzau.edu.cn

收稿日期: 2015-03-02

  修回日期: 2015-06-18

  网络出版日期: 2015-08-03

基金资助

中央高校基本科研业务费专项资金资助项目(编号:2662015BQ005),广东省分子与细胞工程育种团队(编号:2011A020102003)和国家自然科学基金项目(编号:31301226)资助

sgRNA design for the CRISPR/Cas9 system and evaluation of its off-target effects

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  • 1. Key Lab of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China;
    2. Department of Obstetrics and Gynecology, No.161 Hospital of PLA, Wuhan 430010, China

Received date: 2015-03-02

  Revised date: 2015-06-18

  Online published: 2015-08-03

摘要

基于CRISPR/Cas9系统介导的第三代基因组编辑技术,已成功应用于动物、植物和微生物等诸多物种的基因组改造。如何提高CRISPR/Cas9技术的基因组编辑效率和最大限度降低脱靶风险一直是本领域的研究热点,而使用高效且特异的sgRNA(Small guide RNA)是基因组改造成功的关键性因素之一。目前,已有多款针对CRISPR/Cas9技术的sgRNA设计和/或脱靶效应评估软件,但不同的软件各有优缺点。本文重点对16款sgRNA 设计和脱靶效应评估在线和单机版软件的特点进行了阐述,通过制定38项评估指标对不同软件进行了比较分析,最后对11种用于检测基因组编辑效率和脱靶的实验方法,以及如何筛选高效且特异的sgRNA进行了归纳总结。

本文引用格式

谢胜松,张懿,张利生,李广磊,赵长志,倪攀,赵书红 . CRISPR/Cas9系统中sgRNA设计与脱靶效应评估[J]. 遗传, 2015 , 37(11) : 1125 -1136 . DOI: 10.16288/j.yczz.15-093

Abstract

The third generation of CRISPR/Cas9-mediated genome editing technology has been successfully applied to genome modification of various species including animals, plants and microorganisms. How to improve the efficiency of CRISPR/Cas9 genome editing and reduce its off-target effects has been extensively explored in this field. Using sgRNA (Small guide RNA) with high efficiency and specificity is one of the critical factors for successful genome editing. Several software have been developed for sgRNA design and/or off-target evaluation, which have advantages and disadvantages respectively. In this review, we summarize characters of 16 kinds online and standalone software for sgRNA design and/or off-target evaluation and conduct a comparative analysis of these different kinds of software through developing 38 evaluation indexes. We also summarize 11 experimental approaches for testing genome editing efficiency and off-target effects as well as how to screen highly efficient and specific sgRNA.

参考文献

[1] Brouns SJJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJH, Snijders APL, Dickman MJ, Makarova KS, Koonin EV, van der Oost J. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science , 2008, 321(5891): 960-964.
[2] Niu YY, Shen B, Cui YQ, Chen YC, Wang JY, Wang L, Kang Y, Zhao XY, Si W, Li W, Xiang AP, Zhou JK, Guo XJ, Bi Y, Si CY, Hu B, Dong GY, Wang H, Zhou ZM, Li TQ, Tan T, Pu XQ, Wang F, Ji SH, Zhou Q, Huang XX, Ji WZ, Sha JH. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell , 2014, 156(4): 836-843.
[3] Zhou JK, Shen B, Zhang WS, Wang JY, Yang J, Chen L, Zhang N, Zhu K, Xu J, Hu B, Leng QB, Huang XX. One-step generation of different immunodeficient mice with multiple gene modifications by CRISPR/Cas9 mediated genome engineering. Int J Biochem Cell Biol , 2014, 46: 49-55.
[4] Ma YW, Zhang X, Shen B, Lu YD, Chen W, Ma J, Bai L, Huang XX, Zhang LF. Generating rats with conditional alleles using CRISPR/Cas9. Cell Res , 2014, 24(1): 122-125.
[5] Chang NN, Sun CH, Gao L, Zhu D, Xu XF, Zhu XJ, Xiong JW, Xi JJ. Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Res , 2013, 23(4): 465-472.
[6] Hai T, Teng F, Guo RF, Li W, Zhou Q. One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Res , 2014, 24(3): 372-375.
[7] Sato M, Miyoshi K, Nagao Y, Nishi Y, Ohtsuka M, Nakamura S, Sakurai T, Watanabe S. The combinational use of CRISPR/Cas9-based gene editing and targeted toxin technology enables efficientbiallelic knockout of the α-1, 3-galactosyltransferase gene in porcine embryonic fibroblasts. Xenotransplantation , 2014, 21(3): 291-300.
[8] Whitworth KM, Lee K, Benne JA, Beaton BP, Spate LD, Murphy SL, Samuel MS, Mao JD, O'Gorman C, Walters EM, Murphy CN, Driver J, Mileham A, McLaren D, Wells KD, Prather RS. Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos. Biol Reprod , 2014, 91(3): 78.
[9] Zhou XQ, Xin JG, Fan NN, Zou QJ, Huang J, Ouyang Z, Zhao Y, Zhao BT, Liu ZM, Lai SS, Yi XL, Guo L, Esteban MA, Zeng YZ, Yang HQ, Lai LX. Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer. Cell Mol Life Sci , 2015, 72(6): 1175-1184.
[10] Jiang WZ, Zhou HB, Bi HH, Fromm M, Yang B, Weeks DP. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res , 2013, 41(20): e188.
[11] Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell , 2014, 156(5): 935-949.
[12] Barrangou R. RNA events. Cas9 targeting and the CRISPR revolution. Science , 2014, 344(6185): 707-708.
[13] Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature , 2015, 517(7536): 583-588.
[14] Hruscha A, Krawitz P, Rechenberg A, Heinrich V, Hecht J, Haass C, Schmid B. Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish. Development , 2013, 140(24): 4982-4987.
[15] Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I, Sullender M, Ebert BL, Xavier RJ, Root DE. Rational design of highly active sgRNAs for CRISPR- Cas9-mediated gene inactivation. Nat Biotechnol , 2014, 32(12): 1262-1267.
[16] Zhang YL, Ge XL, Yang FY, Zhang LP, Zheng JY, Tan XF, Jin ZB, Qu J, Gu F. Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells. Sci Rep , 2014, 4: 5405.
[17] Farboud B, Meyer BJ. Dramatic Enhancement of Genome Editing by CRISPR/Cas9 Through Improved Guide RNA Design. Genetics , 2015, 199(4): 959-971.
[18] Yu C, Liu YX, Ma TH, Liu K, Xu SH, Zhang Y, Liu HL, La Russa M, Xie M, Ding S, Qi LS. Small Molec
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