CRISPR/Cas9的应用及脱靶效应研究进展

doi:10.16288/j.yczz.15-070

遗传 ›› 2015, Vol. 37 ›› Issue (10): 1003-1010.doi: 10.16288/j.yczz.15-070

CRISPR/Cas9的应用及脱靶效应研究进展

郑武, 谷峰

温州医科大学附属眼视光医院,眼视光学和视觉科学国家重点实验室培育基地,卫生部视觉科学研究重点实验室,温州 325027

收稿日期:2015-02-09 出版日期:2015-10-20 发布日期:2015-10-20
通讯作者: 谷峰,博士,研究员,研究方向：基因修复研究。 E-mail: gufenguw@gmail.com
作者简介:郑武,博士,助理研究员,研究方向：基因修复研究。E-mail: zheng123wu@163.com
基金资助:
国家重点基础研究发展计划(973计划)项目(编号：2013CB967502),国家自然科学基金项目(编号：81201181/H1818),浙江省卫生厅省部共建项目(编号：WKJ2013-2-023)和温州医科大学人才启动项目(编号：QTJ 12011 和KYQD141106)资助

Progress of application and off-target effects of CRISPR/Cas9

Wu Zheng， Feng Gu

State Key Laboratory Cultivating Base of Optometry and Visual Science, Key Laboratory of Vision Science, Ministry of Health, The Eye Hospital of Wenzhou Medical University, Wenzhou 325027 China

Received:2015-02-09 Online:2015-10-20 Published:2015-10-20

摘要/Abstract

摘要： CRISPR/Cas9基因编辑技术在生命科学领域掀起了一场全新的技术革命,该技术可以对基因组特定位点进行靶向编辑,包括缺失、插入、修复等。CRISPR/Cas9比锌指核酸酶 (ZFNs)和转录激活因子样效应物核酸酶(TALENs)技术更易于操作,而且更高效。CRISPR/Cas9系统中的向导RNA(Single guide RNA, sgRNA)是一段与目标DNA片段匹配的RNA序列,指导Cas9蛋白对基因组进行识别。研究发现,设计的sgRNA会与非靶点DNA序列错配,引入非预期的基因突变,即脱靶效应(Off-target effects)。脱靶效应严重制约了CRISPR/Cas9基因编辑技术的广泛应用。为了避免脱靶效应,研究者对影响脱靶效应的因素进行了系统研究并提出了许多降低脱靶效应的方法。文章总结了CRISPR/Cas9系统的应用及脱靶效应研究进展,以期为相关领域的工作提供参考。

关键词: 基因组编辑, CRISPR/Cas9, 脱靶效应, 向导RNA

Abstract: The clustered regulatory interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system mediates genome editing and is revolutionizing genetic researches. Scientists are able to manipulate the gene of interest from any organism with CRISPR/Cas9. Compared with zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) technologies, the CRISPR/Cas9 technology provides an easy and efficient approach to manipulate the genome. In this system, sgRNA (Single guide RNA), a short RNA matching the targeted DNA fragment, guides the CRISPR/Cas9 to interrogate the genome. Because sgRNA can tolerate certain mismatches to the DNA targets and thereby promote undesired off-target mutagenesis, the key limit of this technology is off-target effects. To eliminate the off-target effects, different strategies have been adopted. In this review, we summarize the application of CRISPR/Cas9 and different strategies for addressing off-target effects.

Key words: genome editing, CRISPR/Cas9, off-target effects, sgRNA

郑武, 谷峰. CRISPR/Cas9的应用及脱靶效应研究进展[J]. 遗传, 2015, 37(10): 1003-1010.

Wu Zheng， Feng Gu. Progress of application and off-target effects of CRISPR/Cas9[J]. HEREDITAS(Beijing), 2015, 37(10): 1003-1010.

参考文献

[1] Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science , 2014, 346(6213): 1258096.
[2] Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol , 1987, 169(12): 5429-5433.
[3] Mojica FJ, Díez-Villaseñor C, Soria E, Juez G. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Mol Microbiol , 2000, 36(1): 244-246.
[4] Bolotin A, Quinquis B, Sorokin A, Ehrlich SD. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology , 2005, 151(Pt 8): 2551-2561.
[5] Mojica FJM, Díez-Villaseñor C, García-Martínez J, Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol , 2005, 60(2): 174-182.
[6] Pourcel C, Salvignol G, Vergnaud G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology , 2005, 151(Pt 3): 653-663.
[7] Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct , 2006, 1: 7.
[8] Makarova KS, Aravind L, Wolf YI, Koonin EV. Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems. Biol Direct , 2011, 6: 38.
[9] Makarova KS, Haft DH, Barrangou R, Brouns SJJ, Charpentier E, Horvath P, Moineau S, Mojica FJM, Wolf YI, Yakunin AF, van der Oost J, Koonin EV. Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol , 2011, 9(6): 467-477.
[10] Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao YJ, Pirzada ZA, Eckert MR, Vogel J, Charpentier E. CRISPR RNA maturation by trans -encoded small RNA and host factor RNase III. Nature , 2011, 471(7340): 602-607.
[11] Cong L, Ran FA, Cox D, Lin SL, Barretto R, Habib N, Hsu PD, Wu XB, Jiang WY, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science , 2013, 339(6121): 819-823.
[12] Jiang WY, Bikard D, Cox D, Zhang F, Marraffini LA. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol , 2013, 31(3): 233-239.
[13] Yin H, Xue W, Chen SD, Bogorad RL, Benedetti E, Grompe M, Koteliansky V, Sharp PA, Jacks T, Anderson DG. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat Biotechnol , 2014, 32(6): 551-553.
[14] Wu YX, Liang D, Wang YH, Bai MZ, Tang W, Bao SM, Yan ZQ, Li DS, Li JS. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell , 2013, 13(6): 659-662.
[15] Wu YX, Zhou H, Fan XY, Zhang Y, Zhang M, Wang YH, Xie ZF, Bai MZ, Yin Q, Liang D, Tang W, Liao JY, Zhou CK, Liu WJ, Zhu P, Guo HS, Pan H, Wu CL, Shi HJ, Wu LG, Tang FC, Li JS. Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells. Cell Res , 2015, 25(1): 67-79.
[16] Hu WH, Kaminski R, Yang F, Zhang YG, Cosentino L, Li F, Luo B, Alvarez-Carbonell D, Garcia-Mesa Y, Karn J, Mo XM, Khalili K. RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection. Proc Natl Acad Sci USA , 2014, 111(31): 11461-11466.
[17] Sánchez-Rivera FJ, Papagiannakopoulos T, Romero R, Tammela T, Bauer MR, Bhutkar A, Joshi NS, Subbaraj L, Bronson RT, Xue W, Jacks T. Rapid modelling of cooperating genetic events in cancer through somatic genome editing. Nature , 2014, 516(7531): 428-431.
[18] Findlay GM, Boyle EA, Hause RJ, Klein JC, Shendure J. Saturation edit

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CRISPR/Cas9的应用及脱靶效应研究进展

Progress of application and off-target effects of CRISPR/Cas9

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