CRISPR/Cas9技术的发展及在基因组编辑中的应用

doi:10.13560/j.cnki.biotech.bull.1985.2016.05.007

摘要/Abstract

摘要： CRISPR/Cas9是由细菌和古细菌等微生物中特有的获得性免疫系统发展起来的基因组编辑技术，可以被一段短的RNA引导到复杂基因组中的特定位置，从而对靶标识别切割。该技术可以很容易对几乎所有生物体中的内源基因组DNA序列及其表达产物进行有选择地被编辑或调控，已成为一种热门的基因组编辑工具，正积极推动着从基础生物学到生物技术和医学等方面的发展。介绍CRISPR/Cas9的研究历史、结构和功能以及、精确识别的分子基础，并就其在基因组编辑中的应用进行了较为详尽的综述，以期为从事该领域的科研人员提供参考。

关键词: 核酸内切酶, Cas9, PAM, CRIPR/Cas, 基因组编辑

Abstract: CRISPR/Cas9 is one of novel genome editing technologies developed from the unique prokaryotic acquired immune system in bacteria and archaea，and it can be guided to specific locations within complex genomes by a short RNA，and thus cleavage of the target can be recognized. Using this technology，DNA sequences within the endogenous genome and their expressed products are now easily edited or regulated in virtually any organism of choice. The technology has become the most popular genome editing tool，promoting the development of basic biology，biotechnology and medicine. This paper introduced the research progress on CRISPR/Cas9 from the aspects of development history，structure and function，and the mechanism of precise recognition，also reviewed the application of this new technology in genome editing in detailed，aiming at providing a reference for related researchers.

Key words: engineered endonuclease, Cas9, PAM, CRISPR/Cas, genome editing

张凯丽, 李瑞, 胡桐桐, 徐永杰. CRISPR/Cas9技术的发展及在基因组编辑中的应用[J]. 生物技术通报, 2016, 32(5): 47-60.

ZHANG Kai-li,LI Rui,HU Tong-tong,XU Yong-jie. The Development of CRISPR/Cas9 Technique and Its Applications in Genome Editing[J]. Biotechnology Bulletin, 2016, 32(5): 47-60.

参考文献

[1]Smith J, Grizot S, Arnould S, et al. A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences[J]. Nucleic Acids Res, 2006, 34（22）：e149.
[2]Urnov FD, Miller JC, Lee YL, et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases[J]. Nature, 2005, 435（7042）：646-651.
[3]Boch J, Scholze H, Schornack S, et al. Breaking the code of DNA binding specificity of TAL-type III effectors[J]. Science, 2009, 326（5959）：1509-1512.
[4]Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems[J]. Science, 2013, 339（6121）：819-823.
[5]Mali P, Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9[J]. Science, 2013, 339（6121）：823-826.
[6]Maeder ML, Thibodeau-Beganny S, Osiak A, et al. Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification[J]. Mol Cell, 2008, 2：294-301.
[7]Gonzalez B, Schwimmer LJ, Fuller RP, et al. Modular system for the construction of zinc-finger libraries and proteins[J]. Nat Protoc, 2010, 5（4）：791-810.
[8]Sander JD, Joung JK. CRISPR-Cas systems for editing, regulating and targeting genomes[J]. Nat Biotechnol, 2014, 4：347-355.
[9]Xie K, Minkenberg B, Yang Y. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system[J]. Proc Natl Acad Sci USA, 2015, 11：3570-3575.
[10]Ishino Y, Shinagawa H, Makino K, et al. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product[J]. J Bacteriol, 1987, 169（12）：5429-5433.
[11]Mojica FJ, Diez-Villasenor C, Soria E, et al. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria[J]. Mol Microbiol, 2000, 36（1）：244-246.
[12]Jansen R, Embden JD, Gaastra W, et al. Identification of genes that are associated with DNA repeats in prokaryotes[J]. Mol Microbiol, 2002, 43（6）：1565-1575.
[13]Barrangou R, Fremaux C, Deveau H, et al. CRISPR provides acquired resistance against viruses in prokaryotes[J]. Science, 2007, 315（5819）：1709-1712.
[14]Haft DH, Selengut J, Mongodin EF, et al. A guild of 45 CRISPR-associated（Cas）protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes[J]. PLoS Comput Biol, 2005, 1（6）：e60.
[15]Makarova KS, Haft DH, Barrangou R, et al. Evolution and classification of the CRISPR-Cas systems[J]. Nat Rev Microbiol, 2011, 9（6）：467-477.
[16]Chylinski K, Makarova KS, Charpentier E, et al. Classification and evolution of type II CRISPR-Cas systems[J]. Nucleic Acids Res, 2014, 42（10）：6091-6105.
[17]Bolotin A, Quinquis B, Sorokin A, et al. Clustered regularly interspaced short palindrome repeats（CRISPRs）have spacers of extrachromosomal origin[J]. Microbiology, 2005, 151（Pt 8）：2551-2561.
[18]Mojica FJ, Diez-Villasenor C, Garcia-Martinez J, et al. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements[J]. J Mol Evol, 2005, 2：174-182.
[19]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[J]. Microbiology, 2005, 151（Pt 3）：653-663.
[20]Brouns SJ, Jore MM, Lundgren M, et al. Small CRISPR RNAs guide antiviral defense in prokaryotes[J]. Science, 2008, 321（5891）：960-964.
[21]Marraffini LA, Sontheimer EJ. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA[J]. Science, 2008, 322（5909）：1843-1845.
[22]Hale CR, Zhao P, Olson S, et al. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex[J]. Cell, 2009, 139（5）：945-956.
[23]Deveau H, Barrangou R, Garneau JE, et al. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus[J]. J Bacteriol, 2008, 190（4）：1390-1400.
[24]Horvath P, Coute-Monvoisin AC, Romero DA, et al. Comparative analysis of CRISPR loci in lactic acid bacteria genomes[J]. Int J Food Microbiol, 2009, 131（1）：62-70.
[25]Garneau JE, Dupuis ME, Villion M, et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA[J]. Nature, 2010, 468（7320）：67-71.
[26]Deltcheva E, Chylinski K, Sharma CM, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III[J]. Nature, 2011, 471（7340）：602-607.
[27]Sapranauskas R, Gasiunas G, Fremaux C, et al. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli[J]. Nucleic Acids Res, 2011. 39（21）：9275-9282.
[28]Gasiunas G, Barrangou R, Horvath P, et al. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria[J]. Proc Natl Acad Sci USA, 2012, 109（39）：E2579-2586.
[29]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.
[30]Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9[J]. Science, 2014, 346（6213）：1258096.
[31]Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering[J]. Cell, 2014, 157（6）：1262-1278.
[32] Jinek M, Jiang F, Taylor DW, et al. Structures of Cas9 endonuclea-ses reveal RNA-mediated conformational activation[J]. Science, 2014, 343（6176）：1247997.
[33]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.
[34]Jiang F, Zhou K, Ma L, et al. A Cas9-guide RNA complex preorganized for target DNA recognition[J]. Science, 2015, 348（6242）：1477-1481.
[35]Chylinski K, Le Rhun A, Charpentier E. The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems[J]. RNA Biol, 2013, 10（5）：726-737.
[36]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.
[37]Shah SA, Erdmann S, Mojica FJ, et al. Protospacer recognition motifs：mixed identities and functional diversity[J]. RNA Biol, 2013, 10（5）：891-899.
[38]Sternberg SH, Redding S, Jinek M, et al. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9[J]. Nature, 2014, 507（7490）：62-67.
[39]Hsu PD, Scott DA, Weinstein JA, et al. DNA targeting specificity of RNA-guided Cas9 nucleases[J]. Nat Biotechnol, 2013, 31（9）：827-832.
[40]Jiang W, Bikard D, Cox D, et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems[J]. Nat Biotechnol, 2013, . 31（3）：233-239.
[41] Horvath P, Romero DA, Coute-Monvoisin AC, et al. Diversity, acti-vity, and evolution of CRISPR loci in Streptococcus thermophilus[J]. J Bacteriol, 2008, 190（4）：1401-1412.
[42]Zhang Y, Heidrich N, Ampattu BJ, et al. Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningit-idis[J]. Mol Cell, 2013, 50（4）：488-503.
[43]Hou Z, Zhang Y, Propson NE, et al. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis[J]. Proc Natl Acad Sci USA, 2013, 110（39）：15644-15649.
[44]Niu Y, Shen B, Cui Y, et al. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos[J]. Cell, 2014, 156（4）：836-843.
[45]Kleinstiver BP, Prew MS, Tsai SQ, et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities[J]. Nature, 2015, 523（7561）：481-485.
[46]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.
[47]Mali P, Aach J, Stranges PB, et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering[J]. Nat Biotechnol, 2013, 9：833-838.
[48]Wu X, Scott DA, Kriz AJ, et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells[J]. Nat Biotechnol, 2014, 32（7）：670-676.
[49]Pattanayak V, Lin S, Guilinger JP, et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity[J]. Nat Biotechnol, 2013, 9：839-843.
[50]Gabriel R, Lombardo A, Arens A, et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity[J]. Nat Biotechnol, 2011, 29（9）：816-823.
[51]Kim D, Bae S, Park J, et al. Digenome-seq：genome-wide profiling of CRISPR-Cas9 off-target effects in human cells[J]. Nat Methods, 2015, 12（3）：237-243.
[52]Moreno-Mateos MA, Vejnar CE, Beaudoin JD, et al. CRISPRscan：designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo[J]. Nat Methods, 2015, 12（10）：982-988.
[53]Ran FA, Hsu PD, Lin CY, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity[J]. Cell, 2013, 154（6）：1380-1389.
[54]Fu Y, Sander JD, Reyon D, et al. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs[J]. Nat Biotechnol, 2014, 32（3）：279-284.
[55]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.
[56]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.
[57]Peng J, Zhou Y, Zhu S, et al. High-throughput screens in mammalian cells using the CRISPR-Cas9 system[J]. FEBS J, 2015, 282（1）：2089-2096.
[58]Wiles MV, Qin W, Cheng AW, et al. CRISPR-Cas9-mediated genome editing and guide RNA design[J]. Mamm Genome, 2015, 26（9-10）：501-510.
[59]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.
[60]Smith C, Abalde-Atristain L, He C, et al. Efficient and allele-specific genome editing of disease loci in human iPSCs[J]. Mol Ther, 2015, 23（3）：570-577.
[61]Kearns NA, Genga RM, Enuameh MS, et al. Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells[J]. Development, 2014, 1：219-223.
[62]Li W, Teng F, Li T, et al. Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems[J]. Nat Biotechnol, 2013, 31（8）：684-686.
[63]Yang H, Wang H, Shivalila CS, 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.
[64]Oh B, Hwang S, McLaughlin J, et al. Timely translation during the mouse oocyte-to-embryo transition[J]. Development, 2000, 127（17）：3795-3803.
[65]Zhou Y, Zhu S, Cai C, et al. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells[J]. Nature, 2014, 509（7501）：487-491.
[66]Koike-Yusa H, Li Y, Tan EP, et al. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library[J]. Nat Biotechnol, 2014, 32（3）：267-273.
[67]Shalem O, Sanjana NE, Hartenian E, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells[J]. Science, 2014, 343（6166）：84-87.
[68]Wang T, Wei JJ, Sabatini DM, et al. Genetic screens in human cells using the CRISPR-Cas9 system[J]. Science, 2014, 343（6166）：80-84.
[69]Long L, Guo H, Yao D, et al. Regulation of transcriptionally active genes via the catalytically inactive Cas9 in C. elegans and D. rerio[J]. Cell Res, 2015, 25（5）：638-641.
[70]Gilbert LA, Larson MH, Morsut L, et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes[J]. Cell, 2013, 154（2）：442-451.
[71]Mali P, Esvelt KM, Church GM. Cas9 as a versatile tool for engineering biology[J]. Nat Methods, 2013, 10（10）：957-963.
[72]Perez-Pinera P, Kocak DD, Vockley CM, et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors[J]. Nat Methods, 2013, 10（10）：973-976.
[73] Nihongaki Y, Yamamoto S, Kawano F, et al. CRISPR-Cas9-based photoactivatable transcription system[J]. Chem Biol, 2015, 22（2）：169-174.
[74]Polstein LR, Gersbach CA. A light-inducible CRISPR-Cas9 system for control of endogenous gene activation[J]. Nat Chem Biol, 2015, 11（3）：198-200.
[75]Zetsche B, Volz SE, Zhang F. A split-Cas9 architecture for inducible genome editing and transcription modulation[J]. Nat Biotechnol, 2015, 33（2）：139-142.
[76]Chen B, Gilbert LA, Cimini BA, et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system[J]. Cell, 2013, 155（7）：1479-1491.
[77]Ochiai H, Sugawara T, Yamamoto T. Simultaneous live imaging of the transcription and nuclear position of specific genes[J]. Nucleic Acids Res, 2015, 43（19）：e127.
[78]Long C, McAnally JR, Shelton JM, et al. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA[J]. Science, 2014, 345（6201）：1184-1188.
[79]Liang P, Xu Y, Zhang X, et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes[J]. Protein Cell, 2015, 6（5）：363-372.
[80]Richard GF. Shortening trinucleotide repeats using highly specific endonucleases：a possible approach to gene therapy?[J]. Trends Genet, 2015, 31（4）：177-186.
[81]Vannocci T, Faggianelli N, Zaccagnino S, et al. A new cellular model to follow Friedreich’s ataxia development in a time-resolved way[J]. Dis Model Mech, 2015, 8（7）：711-719.
[82]Lombardo A, Genovese P, Beausejour CM, et al. Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery[J]. Nat Biotechnol, 2007, 25（11）：1298-1306.
[83]Cohen J, Pertsemlidis A, Kotowski IK, et al. Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9[J]. Nat Genet, 2005, 37（2）：161-165.
[84]Musunuru K, Pirruccello JP, Do R, et al. Exome sequencing, ANGPTL3 mutations, and familial combined hypolipidemia[J]. N Engl J Med, 2010, 363（23）：2220-2227.
[85]Hu W, Kaminski R, Yang F, et al. RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection[J]. Proc Natl Acad Sci USA, 2014, 111（31）：11461-11466.
[86]Bi Y, Sun L, Gao D, et al. High-efficiency targeted editing of large viral genomes by RNA-guided nucleases[J]. PLoS Pathog, 2014, 10（5）：e1004090.
[87]Lin SR, Yang HC, Kuo YT, et al. The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo[J]. Mol Ther Nucleic Acids, 2014, 3：e186.
[88]Price AA, Sampson TR, Ratner HK, et al. Cas9-mediated targeting of viral RNA in eukaryotic cells[J]. Proc Natl Acad Sci USA, 2015, 112（19）：6164-6169.
[89]Zhen S, Hua L, Takahashi Y, et al. In vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by CRISPR/Cas9[J]. Biochem Biophys Res Commun, 2014, 450（4）：1422-1426.