CRISPR/Cas9系统介导基因组编辑的研究进展

doi:10.11843/j.issn.0366-6964.2014.10.001

摘要/Abstract

摘要：

由规律成簇间隔短回文重复序列（Clustered regularly interspaced short palindromic repeats，CRISPR）以及CRISPR相关蛋白9（CRISPR-associated protein 9，Cas9），即CRISPR/Cas9系统改造的第3代人工核酸内切酶，与锌指核酸内切酶(Zinc finger endonuclease，ZFN)以及类转录激活因子效应物核酸酶(Transcription activator-like effector nuclease，TALEN)相比，具有构建方法简单快捷、突变效率高、成本低廉、适用范围广等许多优点，已成为一种热门的基因组编辑工具。该技术已成功应用于细菌、人类细胞、斑马鱼、小鼠、猪、食蟹猴以及多种植物的基因组精确修饰，是最具有临床与应用前景的基因治疗技术之一。但是CRISPR/Cas9系统目前也面临着脱靶率高、特异性差的难题，这无疑是CRISPR/Cas9系统应用于基础研究和临床治疗的最大的挑战。本文从CRISPR/Cas9系统的发现、分类、作用机制以及CRISPR/Cas9基因编辑系统在基因定点修饰方面的研究进展进行介绍，希望能够为畜牧领域的科研工作者提供参考。

Abstract:

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) constituted the CRISPR/Cas9 genome editing system，which provides a more efficient way for gene targeting than zinc finger endonuclease (ZFN) and transcription activator-like effector nuclease (TALEN).It is becoming a hot spot in the field of genome editing，and successfully applied to gene targeting of bacteria，human cells，zebra fish，mouse，pig，cynomolgus monkey and several plants.However，the high off-target rate and poor specificity of CRISPR/Cas9 technology hindered its application in medical treatment.Here，the research progress in the structure，classification，function，and mechanism of the CRISPR/Cas9 system is reviewed.

中图分类号:

刘志国. CRISPR/Cas9系统介导基因组编辑的研究进展[J]. 畜牧兽医学报, 2014, 45(10): 1567-1583.

LIU Zhi-guo. Research Progress on CRISPR/Cas9 Mediated Genome Editing[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(10): 1567-1583.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2014.10.001

https://www.xmsyxb.com/CN/Y2014/V45/I10/1567

参考文献

［1］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.
［2］MOJICA F J，FERRER C，JUEZ G，et al.Long stretches of short tandem repeats are present in the largest replicons of the Archaea Haloferax mediterranei and Haloferax volcanii and could be involved in replicon partitioning ［J］.Mol Microbiol，1995，17(1)：85-93.

［3］JANSEN R，EMBDEN J D，GAASTRA W，et al.Identification of genes that are associated with DNA repeats in prokaryotes ［J］.Mol Microbiol，2002，43(6)：1565-1575.
［4］JANSEN R，VAN EMBDEN J D，GAASTRA W，et al.Identification of a novel family of sequence repeats among prokaryotes ［J］.Omics，2002，6(1)：23-33.
［5］BOLOTIN A，QUINQUIS B，SOROKIN A，et al.Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin ［J］.Microbiology，2005，151(8)：2551-2561.
［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 ［J］.Microbiology，2005，151(3)：653-663.
［7］MOJICA F J，DÍEZ-VILLASEÑOR C，GARCÍA-MARTÍNEZ J,et al.Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements ［J］.J Mol Evol，2005，60(2)：174-182.
［8］MAKAROVA K S，GRISHIN N V，SHABALINA S A，et al.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 ［J］.Biol Direct，2006，1(1)：7.
［9］ BARRANGOU R，FREMAUX C，DEVEAU H，et al.CRISPR provides acquired resistance against viruses in prokaryotes ［J］.Science，2007，315(5819)：1709-1712.
［10］MARRAFFINI L A，SONTHEIMER E J.CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA ［J］.Science，2008，322(5909)：1843-1845.
［11］ROUSSEAU C，GONNET M，LE ROMANCER M，et al.CRISPI：a CRISPR interactive database ［J］.Bioinformatics，2009，25(24)：3317-3318.
［12］GRISSA I，VERGNAUD G，POURCEL C.CRISPRFinder：a web tool to identify clustered regularly interspaced short palindromic repeats ［J］.Nucleic Acids Res，2007，35(suppl 2)：W52-W57.
［13］GRISSA I，VERGNAUD G，POURCEL C.The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats ［J］.BMC Bioinformatics，2007，8(1)：172.
［14］DEVEAU H，GARNEAU J E，MOINEAU S.CRISPR/Cas system and its role in phage-bacteria interactions ［J］.Annu Rev Microbiol，2010，64：475-493.
［15］KUNIN V，SOREK R，HUGENHOLTZ P.Evolutionary conservation of sequence and secondary structures in CRISPR repeats ［J］.Genome Biol，2007，8(4)：R61.
［16］GODDE J S，BICKERTON A.The repetitive DNA elements called CRISPRs and their associated genes：evidence of horizontal transfer among prokaryotes ［J］. J Mol Evol，2006，62(6)：718-729.
［17］HAFT D H，SELENGUT J，MONGODIN E F，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.
［18］WIEDENHEFT B，ZHOU K，JINEK M，et al.Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated genome defense ［J］.Structure，2009，17(6)：904-912.
［19］HAN D，KRAUSS G.Characterization of the endonuclease SSO2001 from Sulfolobus solfataricus P2 ［J］.FEBS Lett, 2009，583(4)：771-776.
［20］BHAYA D，DAVISON M，BARRANGOU R.CRISPR-Cas systems in bacteria and archaea：versatile small RNAs for adaptive defense and regulation ［J］.Annu Rev Genet，2011，45：273-297.
［21］VAN DER OOST J，JORE M M，WESTRA E R，et al.CRISPR-based adaptive and heritable immunity in prokaryotes ［J］. Trends Biochem Sci，2009，34(8)：401-407.
［22］BELOGLAZOVA N，BROWN G，ZIMMERMAN M D，et al.A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats ［J］.J Biol Chem，2008，283(29)：20361-20371.
［23］MAKAROVA K S，HAFT D H，BARRANGOU R，et al.Evolution and classification of the CRISPR–Cas systems ［J］.Nat Rev Microbiol，2011，9(6)：467-477.
［24］AGARI Y，SAKAMOTO K，TAMAKOSHI M，et al.Transcription profile of thermus thermophilus CRISPR systems after phage infection ［J］. J Mol Biol，2010，395(2)：270-281.
［25］POUGACH K，SEMENOVA E，BOGDANOVA E，et al.Transcription，processing and function of CRISPR cassettes in Escherichia coli ［J］.Mol Microbiol，2010，77(6)：1367-1379.
［26］BROUNS S J J，JORE M M，LUNDGREN M，et al.Small CRISPR RNAs guide antiviral defense in prokaryotes ［J］.Science，2008，321(5891)：960-964.
［27］MARRAFFINI L A，SONTHEIMER E J.Self versus non-self discrimination during CRISPR RNA-directed immunity ［J］.Nature，2010，463(7280)：568-571.
［28］MOJICA F J，DIEZ-VILLASENOR C，GARCIA-MARTINEZ J，et al.Short motif sequences determine the targets of the prokaryotic CRISPR defence system ［J］.Microbiology，2009，155(3)：733-740.
［29］JINEK M，JIANG F，TAYLOR D W，et al.Structures of Cas9 endonucleases reveal RNA-mediated conformational activation ［J］. Science，2014，343(6176)：1247997.
［30］SINKUNAS T，GASIUNAS G，FREMAUX C，et al.Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system ［J］.EMBO J，2011，30(7)：1335-1342.
［31］DELTCHEVA E，CHYLINSKI K，SHARMA C M，et al.CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III ［J］. Nature，2011，471(7340)：602-607.
［32］ANANTHARAMAN V，IYER L M，ARAVIND L.Presence of a classical RRM-fold palm domain in Thg1-type 3′-5′nucleic acid polymerases and the origin of the GGDEF and CRISPR polymerase domains ［J］.Biol Direct，2010，5：43.
［33］HALE C R，ZHAO P，OLSON S，et al.RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex ［J］.Cell, 2009，139(5)：945-956.
［34］GASIUNAS G，SIKSNYS V.RNA-dependent DNA endonuclease Cas9 of the CRISPR system：Holy Grail of genome editing? ［J］.Trends Microbiol，2013，21(11)：1-6.
［35］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.
［36］MALI P，YANG L，ESVELT K M，et al.RNA-guided human genome engineering via Cas9 ［J］.Science，2013，339(6121)：823-826.
［37］CONG L，RAN F A，COX D，et al.Multiplex genome engineering using CRISPR/Cas systems ［J］.Science，2013，339(6121)：819-823.
［38］CHO S W，KIM S，KIM J M，et al.Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease ［J］.Nat Biotechnol，2013，31(3)：230-232.
［39］HWANG W Y，FU Y，REYON D，et al.Efficient genome editing in zebrafish using a CRISPR-Cas system ［J］.Nat Biotechnol，2013，31(3)：227-229.
［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］CHANG N，SUN C，GAO L，et al.Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos ［J］.Cell Res，2013，23(4)：465-472.
［42］XIAO A，WANG Z，HU Y，et al.Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish ［J］.Nucleic Acids Res，2013，41(14)：e141.
［43］JAO L E，WENTE S R，CHEN W.Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system ［J］.Proceedings of the National Academy of Sciences，2013，110(34)：13904-13909.
［44］SHEN B，ZHANG J，WU H，et al.Generation of gene-modified mice via Cas9/RNA-mediated gene targeting ［J］.Cell Res，2013，23(5)：720-723.
［45］WANG H，YANG H，SHIVALILA C S，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.
［46］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.
［47］FURTHERMORE A，RNA C.Heritable gene targeting in the mouse and rat using a CRISPR-Cas system ［J］.Nat Biotechnol，2013，31(8)：681-683.
［48］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.
［49］HAI T，TENG F，GUO R，et al.One-step generation of knockout pigs by zygote injection of CRISPR/Cas system ［J］.Front Agr Sci Eng，2014，1(1)：2-5.
［50］LI X，YANG Y，BU L，et al.Rosa26-targeted swine models for stable gene over-expression and Cre-mediated lineage tracing ［J］.Cell Res，2014，24(4)：501-504.
［51］QI L S，LARSON M H，GILBERT L A，et al.Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression ［J］.Cell，2013，152(5)：1173-1183.
［52］BIKARD D，JIANG W，SAMAI P，et al.Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system ［J］.Nucleic Acids Res，2013，41(15)：7429-7437.
［53］GILBERT L A，MORSUT M，LEONARDO H L，et al.CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes ［J］.Cell，2013，154(2)：442-451.
［54］MAEDER M L，LINDER S J，CASCIO V M，et al.CRISPR RNA-guided activation of endogenous human genes ［J］.Nat Methods，2013，10(10)：977-979.
［55］PEREZ-PINERA P，KOCAK D D，VOCKLEY C M，et al.RNA-guided gene activation by CRISPR- Cas9-based transcription factors ［J］.Nat Methods，2013，10(10)：973-976.
［56］KONERMANN S，BRIGHAM M D，TREVINO A，et al.Optical control of mammalian endogenous transcription and epigenetic states ［J］. Nature，2013，500(7463)：472-476.
［57］HEN B，ZHANG J，WU H，et al.Generation of gene-modified mice via Cas9/RNA-mediated gene targeting ［J］.Cell Res，2013，23(5)：720-723.
［58］BASSETT A R，TIBBIT C，PONTING C P，et al.Highly efficient targeted mutagenesis of drosophila with the CRISPR/Cas9 system ［J］.Cell Reports，2013，4(1)：220-228.
［59］FRIEDLAND A E，TZUR Y B，ESVELT K M，et al.Heritable genome editing in C.elegans via a CRISPR-Cas9 system ［J］.Nat Methods，2013，10(8)：741-743.
［60］HSU P D，SCOTT D A，WEINSTEIN J A，et al.DNA targeting specificity of RNA-guided Cas9 nucleases ［J］.Nat Biotechnol，2013，31(9)：827-832.
［61］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.
［62］JINEK M，EAST A，CHENG A，et al.RNA-programmed genome editing in human cells ［J］.eLife，2013，2(3)：e00471.
［63］FU Y，FODEN J A，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.
［64］MALI P，AACH J，STRANGES P B，et al.CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering ［J］.Nat Biotechnol，2013，31(9)：833-838.
［65］PATTANAYAK V，LIN S，GUILINGER J P，et al.High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity ［J］.Nat Biotechnol，2013，31(9)：839-843.
［66］RAN F A，HSU P D，LIN C Y，et al.Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity ［J］.Cell，2013，154(6)：1380-1389.
［67］KOIKE-YUSA H，LI Y，TAN E P，et al.Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library ［J］.Nat Biotechnol，2013，32(3)：267-273.
［68］WANG T，WEI J J，SABATINI D M，et al.Genetic screens in human cells using the CRISPR-Cas9 system ［J］.Science，2014，343(6166)：80-84.
［69］SHALEM O，SANJANA N E，HARTENIAN E，et al.Genome-scale CRISPR-Cas9 knockout screening in human cells ［J］.Science，2014，343(6166)：84-87.