干酪乳杆菌CRISPR基因座分析
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摘要
【目的】目前基于酿脓链球菌(Streptococcus pyogenes)spCas9为核心的CRISPR/Cas9基因编辑系统在乳酸菌上的应用受到很多限制,亟待开发适合于乳酸菌的基因编辑系统。对6株干酪乳杆菌(Lactobacillus casei)的CRISPR系统进行深入分析,并预测激活干酪乳杆菌自身Cas9蛋白所识别的PAM序列,为开发适用于乳酸菌的CRISPR/lcCas9基因编辑系统奠定基础。【方法】以已完成全基因组测序的6株干酪乳杆菌为研究对象,利用生物信息学方法对其CRISPR系统进行深入分析,重点对不同菌株的CRISPR系统结构进行解析,并且对Cas蛋白以及spacer的同源性进行分析,最后对CRISPR区重复序列的二级结构以及Cas9蛋白识别的PAM序列进行预测。【结果】6株干酪乳杆菌CRISPR系统具有相似的结构,均具有特征性的Cas9蛋白,并且Cas基因序列保守。预测到tracrRNA位于Cas9和Cas1之间,重复序列可以形成茎部长达7个碱基的二级结构。根据CRISPR的间隔区序列,6株干酪乳杆菌可被分为3个基因型,将间隔区逐一进行blast比对,结果表明6个间隔区比对上14个来源不同的原间隔序列,这些间隔序列均来源于不同质粒。干酪乳杆菌lcCas9蛋白识别PAM序列的1、3位碱基偏好T/C、A/C,2、4位碱基对G、A的偏好性比较大。【结论】6株干酪乳杆菌CRISPR系统均为type-ⅡA型,Cas序列和重复序列高度保守。DR序列可以形成稳定的二级结构,TGMA为干酪乳杆菌Cas9蛋白高效识别的PAM序列。
【Objective】The application of the CRISPR/Cas9 gene editing system based on Streptococcus pyogenes spCas9 has restriction on lactic acid bacteria at present. It is urgent to develop a suitable gene editing system for lactic acid bacteria. In this study, we analysed the CRISPR system of Lactobacillus casei in depth, and then predicted the PAM sequence to activate its Cas9 protein. Our study provided the experimental foundation for the development of the CRISPR/lcCas9 gene editing system for lactic acid bacteria 【Method】In this study, six strains of whole genome-sequenced L. casei were used as research object. Bioinformatics was used to analyze the CRISPR system. Cas protein structure, CRISPR system and homology of space were analysed. At the end, the second structure of the CRISPR repeat and PAM sequence recognized by Cas9 protein were predicted. 【Result】The CRISPR system of L. casei had similar structures characteristic of Cas9 protein, and the Cas gene were conserved. It was predicted that the tracrRNA was located between Cas9 and Cas1, and the repeat sequence could form a secondary structure with a stem length of seven bases. According to the sequence characteristics of CRISPR spacers, six Lactobacillus casei could be divided into three genotypes. Then the spacer sequences were blasted one by one. The results showed that the six spacers aligned 14 original source sequences with different origins, and these spacer sequences were all derived from different plasmids. The PAM sequence recognized by L. casei lcCas9 protein preferred T/C, A/C at the 1st and 3rd bases. The 2nd and 4th bases had greater preferences for G and A.【Conclusion】The CRISPR system of six strains of L. casei were all type-IIA. Consequently, the Cas gene and repeat sequences were highly conserved. The DR sequence formed a stable secondary structure, while TGMA as a PAM sequence was effectively identified by the Cas9 proetein of L. casei.
【Objective】The application of the CRISPR/Cas9 gene editing system based on Streptococcus pyogenes spCas9 has restriction on lactic acid bacteria at present. It is urgent to develop a suitable gene editing system for lactic acid bacteria. In this study, we analysed the CRISPR system of Lactobacillus casei in depth, and then predicted the PAM sequence to activate its Cas9 protein. Our study provided the experimental foundation for the development of the CRISPR/lcCas9 gene editing system for lactic acid bacteria 【Method】In this study, six strains of whole genome-sequenced L. casei were used as research object. Bioinformatics was used to analyze the CRISPR system. Cas protein structure, CRISPR system and homology of space were analysed. At the end, the second structure of the CRISPR repeat and PAM sequence recognized by Cas9 protein were predicted. 【Result】The CRISPR system of L. casei had similar structures characteristic of Cas9 protein, and the Cas gene were conserved. It was predicted that the tracrRNA was located between Cas9 and Cas1, and the repeat sequence could form a secondary structure with a stem length of seven bases. According to the sequence characteristics of CRISPR spacers, six Lactobacillus casei could be divided into three genotypes. Then the spacer sequences were blasted one by one. The results showed that the six spacers aligned 14 original source sequences with different origins, and these spacer sequences were all derived from different plasmids. The PAM sequence recognized by L. casei lcCas9 protein preferred T/C, A/C at the 1st and 3rd bases. The 2nd and 4th bases had greater preferences for G and A.【Conclusion】The CRISPR system of six strains of L. casei were all type-IIA. Consequently, the Cas gene and repeat sequences were highly conserved. The DR sequence formed a stable secondary structure, while TGMA as a PAM sequence was effectively identified by the Cas9 proetein of L. casei.
引文
[1]COBB R E,WANG Y,ZHAO H.High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system.ACS Synthetic Biology,2015,4(6):723-728.
[2]JIANG Y,CHEN B,DUAN C,SUN B,YANG J,YANG S.Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system.Applied and Environmental Microbiology,2015,81(7):2506-2514.
[3]PYNE M E,MOO-YOUNG M,CHUNG D A,CHOU C P.Coupling the CRISPR/Cas9 system with lambda red recombineering enables simplified chromosomal gene replacement in Escherichia coli.Applied and Environmental Microbiology,2015,81(15):5103-5114.
[4]陈冲,宋丽菊,齐盼盼,王敏,冯盨,张杰,赵建.干酪乳杆菌在乳制品中的应用研究进展.食品与发酵科技,2015,51(4):88-91.CHEN C,SONG L J,QI P P,WANG M,FENG W,ZHANG J,ZHAOJ.Progress in the application of Lactobacillus casei in dairy products.Food and Fermentation Technology,2015,51(4):88-91.(in Chinese)
[5]KOEBNICK C,WAGNER I,LEITZMANN P,STERN U,ZUNFT HJ.Probiotic beverage containing Lactobacillus casei Shirota improves gastrointestinal symptoms in patients with chronic constipation.Canadian Journal of Gastroenterology,2016,17(11):655-659.
[6]AKOGLU B,LOYTVED A,NUIDING H,ZEUZEM S,FAUST D.Probiotic Lactobacillus casei Shirota improves kidney function,inflammation and bowel movements in hospitalized patients with acute gastroenteritis-A prospective study.Journal of Functional Foods,2015,17:305-313.
[7]MAKAROVA K S,WOLF Y I,KOONIN E V.Comparative genomics of defense systems in archaea and bacteria.Nucleic Acids Research,2013,41(8):4360-4377.
[8]BOLOTIN A,QUINQUIS B,SOROKIN A,EHRLICH S D.Clustered regularly interspaced short palindrome repeats(CRISPRs)have spacers of extrachromosomal origin.Microbiology,2005,151(Pt 8):2551-2561.
[9]VISWANATHAN P,MURPHY K,JULIEN B,GARZA A G,KROOSL.Regulation of dev,an operon that includes genes essential for Myxococcus xanthus development and CRISPR-associated genes and repeats.Journal of Bacteriology,2007,189(10):3738-3750.
[10]KARVELIS T,GASIUNAS G,MIKSYS A,BARRANGOU R,HORVATH P,SIKSNYS V.CrRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus.RNABiology,2013,10(5):841-851.
[11]DELTCHEVA E,CHYLINSKI K,SHARMA C M,GONZALES K,CHAO Y,PIRZADA Z A,ECKERT M R,VOGEL J,CHARPENTIERE.CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.Nature,2011,471(7340):602-607.
[12]VAN DER OOST J,JORE M M,WESTRA E R,LUNDGREN M,BROUNS S J.CRISPR-based adaptive and heritable immunity in prokaryotes.Trends in Biochemical Sciences,2009,34(8):401-407.
[13]FUJII W,ONUMA A,SUGIURA K,NAITO K.Efficient generation of genome-modified mice via offset-nicking by CRISPR/Cas system.Biochemical&Biophysical Research Communications,2014,445(4):791-794.
[14]AIDA T,CHIYO K,USAMI T,ISHIKUBO H,IMAHASHI R,WADA Y,TANAKA K F,SAKUMA T,YAMAMOTO T,TANAKA K.Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice.Genome Biology,2015,16(1):1-11.
[15]NAKAGAWA Y,SAKUMA T,SAKAMOTO T,OHMURAYA M,NAKAGATA N,YAMAMOTO T.Production of knockout mice by DNA microinjection of various CRISPR/Cas9 vectors into freezethawed fertilized oocytes.BMC Biotechnology,2015,15(1):33.
[16]MA Y W,ZHANG X,SHEN B,LU Y D,CHEN W,MA J,BAI L,HUANG X X,ZHANG L F.Generating rats with conditional alleles using CRISPR/Cas9.Cell Research,2014,24(1):122-125.
[17]VARSHNEY G K,PEI W,LAFAVE M C,IDOL J,XU L,GALLARDO V,CARRINGTON B,BISHOP K,JONES M,LI M.High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9.Genome Research,2015,25(7):1030-1042.
[18]AUER T O,DUROURE K,DE CIAN A,CONCORDET J P,DELBENE F.Highly efficient CRISPR/Cas9 mediated knock-in in zebrafish by homology-independent DNA repair.Genome Research,2014,24(1):142.
[19]SHEN Z F,ZHANG X L,CHAI Y P,ZHU Z W,YI P S,FENG G X,LI W,OU G S.Conditional knockouts generated by engineered CRISPR-Cas9 endonuclease reveal the roles of coronin in C.elegans neural development.Developmental Cell,2014,30(5):625-636.
[20]MIAO J,GUO D S,ZHANG J Z,HUANG Q P,QIN G J,ZHANG X,WAN J M,GU H Y,QU L J.Targeted mutagenesis in rice using CRISPR-Cas system.Cell Research,2013,23(10):1233-1236.
[21]FENG Z Y,MAO Y F,XU N F,ZHANG B T,WEI P L,YANG D L,WANG Z,ZHANG Z J,ZHENG R,YANG L,ZENG L,LIU X D,ZHU J K.Multigeneration analysis reveals the inheritance,specificity,and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis.Proceedings of the National Academy of Sciences of the United States of America,2014,111(12):4632-4637.
[22]LUN C,BIKARD D.Consequences of Cas9 cleavage in the chromosome of Escherichia coli.Nucleic Acids Research,2016,44(9):4243-4251.
[23]JI W Y,LEE D,WONG E,DADLANI P,DINH D,HUANG V,KEARNS K,TENG S,CHEN S,HALIBURTON J,HALIBURTON J,HEIMBERG G,HEINEIKE B,RAMASUBRAMANIAN A,STEVENS T,HELMKE K J,ZEPEDA V,QI L S,LIM W A.Specific gene repression by CRISPRi system transferred through bacterial conjugation.Acs Synthetic Biology,2014,3(12):929-931.
[24]JINEK M,CHYLINSKI K,FONFARA I,HAUER M,DOUDNA J A,CHARPENTIER E.A programmable dual-RNA-guided DNAendonuclease in adaptive bacterial immunity.Science,2012,337(6096):816-821.
[25]JIANG W,BIKARD D,COX D,ZHANG F,MARRAFFINI L A.RNA-guided editing of bacterial genomes using CRISPR-Cas systems.Nature Biotechnology,2013,31(3):233-239.
[26]BURGESS D J.Technology:A CRISPR genome-editing tool.Nature Reviews Genetics,2013,14(2):80.
[27]OH J H,VAN PIJKEREN J P.CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri.Nucleic Acids Research,2014,42(17):e131.
[28]BIDART G N,RODRIGUEZ-DIAZ J,MONEDERO V,YEBRA M J.A unique gene cluster for the utilization of the mucosal and human milk-associated glycans galacto-N-biose and lacto-N-biose in Lactobacillus casei.Molecular Microbiology,2014,93(3):521-538.
[29]CHYLINSKI K,LE RHUN A,CHARPENTIER E.The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems.RNA Biology,2013,10(5):726-737.
[30]HORVATH P,BARRANGOU R.CRISPR/Cas,the immune system of bacteria and archaea.Science,2010,327(5962):167-170.
[31]BARRANGOU R,FREMAUX C,DEVEAU H,RICHARDS M,BOYAVAL P,MOINEAU S,ROMERO D A,HORVATH P.CRISPRprovides acquired resistance against viruses in prokaryotes.Science,2007,315(5819):1709-1712.
[32]GARNEAU J E,DUPUIS M E,VILLION M,ROMERO D A,BARRANGOU R,BOYAVAL P,FREMAUX C,HORVATH P,MAGADAN A H,MOINEAU S.The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA.Nature,2010,468(7320):67-71.
[33]SAPRANAUSKAS R,GASIUNAS G,FREMAUX C,BARRANGOU R,HORVATH P,SIKSNYS V.The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli.Nucleic Acids Research,2011,39(21):9275-9282.
[34]MARRAFFINI L A,SONTHEIMER E J.CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA.Science,2008,322(5909):1843.
[35]BARRANGOU R,HORVATH P.A decade of discovery:CRISPRfunctions and applications.Nature Microbiology,2017,2:17092.
[36]BRINER A E,LUGLI G A,MILANI C,DURANTI S,TURRONI F,GUEIMONDE M,MARGOLLES A,VAN SINDEREN D,VENTURA M,BARRANGOU R.Occurrence and diversity of CRISPR-Cas systems in the genus bifidobacterium.PLoS ONE,2015,10(7):e0133661.
[37]DEVEAU H,BARRANGOU R,GARNEAU J E,LABONTE J,FREMAUX C,BOYAVAL P,ROMERO D A,HORVATH P,MOINEAU S.Phage response to CRISPR-encoded resistance in Streptococcus thermophilus.Journal of Bacteriology,2008,190(4):1390-1400.
[38]PAEZ-ESPINO D,MOROVIC W,SUN C L,THOMAS B C,UEDAK,STAHL B,BARRANGOU R,BANFIELD J F.Strong bias in the bacterial CRISPR elements that confer immunity to phage.Nature Communications,2013,4:1430.
[39]GASIUNAS G,BARRANGOU R,HORVATH P,SIKSNYS V.Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.Proceedings of the National Academy of Sciences of the United States of America,2012,109(39):E2579-E2586.
[40]MOJICA F J,DIEZ-VILLASENOR C,GARCIA-MARTINEZ J,ALMENDROS C.Short motif sequences determine the targets of the prokaryotic CRISPR defence system.Microbiology,2009,155(Pt 3):733-740.
[41]HSU P D,SCOTT D A,WEINSTEIN J A,RAN F A,KONERMANNS,AGARWALA V,LI Y,FINE E J,WU X,SHALEM O,CRADICKT J,MARRAFFINI L A,BAO G,ZHANG F.DNA targeting specificity of RNA-guided Cas9 nucleases.Nature Biotechnology,2013,31(9):827-832.
[42]ZHANG Y,GE X,YANG F,ZHANG L,ZHENG J,TAN X,JIN Z B,QU J,GU F.Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells.Scientific Reports,2014,4:5405.
[43]RAN F A,CONG L,YAN W X,SCOTT D A,GOOTENBERG J S,KRIZ A J,ZETSCHE B,SHALEM O,WU X,MAKAROVA K S,KOONIN E V,SHARP P A,ZHANG F.In vivo genome editing using Staphylococcus aureus Cas9.Nature,2015,520(7546):186-191.
[44]KLEINSTIVER B P,PREW M S,TSAI S Q,NGUYEN N T,TOPKAR V V,ZHENG Z,JOUNG J K.Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAMrecognition.Nature Biotechnology,2015,33(12):1293-1298.
[2]JIANG Y,CHEN B,DUAN C,SUN B,YANG J,YANG S.Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system.Applied and Environmental Microbiology,2015,81(7):2506-2514.
[3]PYNE M E,MOO-YOUNG M,CHUNG D A,CHOU C P.Coupling the CRISPR/Cas9 system with lambda red recombineering enables simplified chromosomal gene replacement in Escherichia coli.Applied and Environmental Microbiology,2015,81(15):5103-5114.
[4]陈冲,宋丽菊,齐盼盼,王敏,冯盨,张杰,赵建.干酪乳杆菌在乳制品中的应用研究进展.食品与发酵科技,2015,51(4):88-91.CHEN C,SONG L J,QI P P,WANG M,FENG W,ZHANG J,ZHAOJ.Progress in the application of Lactobacillus casei in dairy products.Food and Fermentation Technology,2015,51(4):88-91.(in Chinese)
[5]KOEBNICK C,WAGNER I,LEITZMANN P,STERN U,ZUNFT HJ.Probiotic beverage containing Lactobacillus casei Shirota improves gastrointestinal symptoms in patients with chronic constipation.Canadian Journal of Gastroenterology,2016,17(11):655-659.
[6]AKOGLU B,LOYTVED A,NUIDING H,ZEUZEM S,FAUST D.Probiotic Lactobacillus casei Shirota improves kidney function,inflammation and bowel movements in hospitalized patients with acute gastroenteritis-A prospective study.Journal of Functional Foods,2015,17:305-313.
[7]MAKAROVA K S,WOLF Y I,KOONIN E V.Comparative genomics of defense systems in archaea and bacteria.Nucleic Acids Research,2013,41(8):4360-4377.
[8]BOLOTIN A,QUINQUIS B,SOROKIN A,EHRLICH S D.Clustered regularly interspaced short palindrome repeats(CRISPRs)have spacers of extrachromosomal origin.Microbiology,2005,151(Pt 8):2551-2561.
[9]VISWANATHAN P,MURPHY K,JULIEN B,GARZA A G,KROOSL.Regulation of dev,an operon that includes genes essential for Myxococcus xanthus development and CRISPR-associated genes and repeats.Journal of Bacteriology,2007,189(10):3738-3750.
[10]KARVELIS T,GASIUNAS G,MIKSYS A,BARRANGOU R,HORVATH P,SIKSNYS V.CrRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus.RNABiology,2013,10(5):841-851.
[11]DELTCHEVA E,CHYLINSKI K,SHARMA C M,GONZALES K,CHAO Y,PIRZADA Z A,ECKERT M R,VOGEL J,CHARPENTIERE.CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.Nature,2011,471(7340):602-607.
[12]VAN DER OOST J,JORE M M,WESTRA E R,LUNDGREN M,BROUNS S J.CRISPR-based adaptive and heritable immunity in prokaryotes.Trends in Biochemical Sciences,2009,34(8):401-407.
[13]FUJII W,ONUMA A,SUGIURA K,NAITO K.Efficient generation of genome-modified mice via offset-nicking by CRISPR/Cas system.Biochemical&Biophysical Research Communications,2014,445(4):791-794.
[14]AIDA T,CHIYO K,USAMI T,ISHIKUBO H,IMAHASHI R,WADA Y,TANAKA K F,SAKUMA T,YAMAMOTO T,TANAKA K.Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice.Genome Biology,2015,16(1):1-11.
[15]NAKAGAWA Y,SAKUMA T,SAKAMOTO T,OHMURAYA M,NAKAGATA N,YAMAMOTO T.Production of knockout mice by DNA microinjection of various CRISPR/Cas9 vectors into freezethawed fertilized oocytes.BMC Biotechnology,2015,15(1):33.
[16]MA Y W,ZHANG X,SHEN B,LU Y D,CHEN W,MA J,BAI L,HUANG X X,ZHANG L F.Generating rats with conditional alleles using CRISPR/Cas9.Cell Research,2014,24(1):122-125.
[17]VARSHNEY G K,PEI W,LAFAVE M C,IDOL J,XU L,GALLARDO V,CARRINGTON B,BISHOP K,JONES M,LI M.High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9.Genome Research,2015,25(7):1030-1042.
[18]AUER T O,DUROURE K,DE CIAN A,CONCORDET J P,DELBENE F.Highly efficient CRISPR/Cas9 mediated knock-in in zebrafish by homology-independent DNA repair.Genome Research,2014,24(1):142.
[19]SHEN Z F,ZHANG X L,CHAI Y P,ZHU Z W,YI P S,FENG G X,LI W,OU G S.Conditional knockouts generated by engineered CRISPR-Cas9 endonuclease reveal the roles of coronin in C.elegans neural development.Developmental Cell,2014,30(5):625-636.
[20]MIAO J,GUO D S,ZHANG J Z,HUANG Q P,QIN G J,ZHANG X,WAN J M,GU H Y,QU L J.Targeted mutagenesis in rice using CRISPR-Cas system.Cell Research,2013,23(10):1233-1236.
[21]FENG Z Y,MAO Y F,XU N F,ZHANG B T,WEI P L,YANG D L,WANG Z,ZHANG Z J,ZHENG R,YANG L,ZENG L,LIU X D,ZHU J K.Multigeneration analysis reveals the inheritance,specificity,and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis.Proceedings of the National Academy of Sciences of the United States of America,2014,111(12):4632-4637.
[22]LUN C,BIKARD D.Consequences of Cas9 cleavage in the chromosome of Escherichia coli.Nucleic Acids Research,2016,44(9):4243-4251.
[23]JI W Y,LEE D,WONG E,DADLANI P,DINH D,HUANG V,KEARNS K,TENG S,CHEN S,HALIBURTON J,HALIBURTON J,HEIMBERG G,HEINEIKE B,RAMASUBRAMANIAN A,STEVENS T,HELMKE K J,ZEPEDA V,QI L S,LIM W A.Specific gene repression by CRISPRi system transferred through bacterial conjugation.Acs Synthetic Biology,2014,3(12):929-931.
[24]JINEK M,CHYLINSKI K,FONFARA I,HAUER M,DOUDNA J A,CHARPENTIER E.A programmable dual-RNA-guided DNAendonuclease in adaptive bacterial immunity.Science,2012,337(6096):816-821.
[25]JIANG W,BIKARD D,COX D,ZHANG F,MARRAFFINI L A.RNA-guided editing of bacterial genomes using CRISPR-Cas systems.Nature Biotechnology,2013,31(3):233-239.
[26]BURGESS D J.Technology:A CRISPR genome-editing tool.Nature Reviews Genetics,2013,14(2):80.
[27]OH J H,VAN PIJKEREN J P.CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri.Nucleic Acids Research,2014,42(17):e131.
[28]BIDART G N,RODRIGUEZ-DIAZ J,MONEDERO V,YEBRA M J.A unique gene cluster for the utilization of the mucosal and human milk-associated glycans galacto-N-biose and lacto-N-biose in Lactobacillus casei.Molecular Microbiology,2014,93(3):521-538.
[29]CHYLINSKI K,LE RHUN A,CHARPENTIER E.The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems.RNA Biology,2013,10(5):726-737.
[30]HORVATH P,BARRANGOU R.CRISPR/Cas,the immune system of bacteria and archaea.Science,2010,327(5962):167-170.
[31]BARRANGOU R,FREMAUX C,DEVEAU H,RICHARDS M,BOYAVAL P,MOINEAU S,ROMERO D A,HORVATH P.CRISPRprovides acquired resistance against viruses in prokaryotes.Science,2007,315(5819):1709-1712.
[32]GARNEAU J E,DUPUIS M E,VILLION M,ROMERO D A,BARRANGOU R,BOYAVAL P,FREMAUX C,HORVATH P,MAGADAN A H,MOINEAU S.The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA.Nature,2010,468(7320):67-71.
[33]SAPRANAUSKAS R,GASIUNAS G,FREMAUX C,BARRANGOU R,HORVATH P,SIKSNYS V.The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli.Nucleic Acids Research,2011,39(21):9275-9282.
[34]MARRAFFINI L A,SONTHEIMER E J.CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA.Science,2008,322(5909):1843.
[35]BARRANGOU R,HORVATH P.A decade of discovery:CRISPRfunctions and applications.Nature Microbiology,2017,2:17092.
[36]BRINER A E,LUGLI G A,MILANI C,DURANTI S,TURRONI F,GUEIMONDE M,MARGOLLES A,VAN SINDEREN D,VENTURA M,BARRANGOU R.Occurrence and diversity of CRISPR-Cas systems in the genus bifidobacterium.PLoS ONE,2015,10(7):e0133661.
[37]DEVEAU H,BARRANGOU R,GARNEAU J E,LABONTE J,FREMAUX C,BOYAVAL P,ROMERO D A,HORVATH P,MOINEAU S.Phage response to CRISPR-encoded resistance in Streptococcus thermophilus.Journal of Bacteriology,2008,190(4):1390-1400.
[38]PAEZ-ESPINO D,MOROVIC W,SUN C L,THOMAS B C,UEDAK,STAHL B,BARRANGOU R,BANFIELD J F.Strong bias in the bacterial CRISPR elements that confer immunity to phage.Nature Communications,2013,4:1430.
[39]GASIUNAS G,BARRANGOU R,HORVATH P,SIKSNYS V.Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.Proceedings of the National Academy of Sciences of the United States of America,2012,109(39):E2579-E2586.
[40]MOJICA F J,DIEZ-VILLASENOR C,GARCIA-MARTINEZ J,ALMENDROS C.Short motif sequences determine the targets of the prokaryotic CRISPR defence system.Microbiology,2009,155(Pt 3):733-740.
[41]HSU P D,SCOTT D A,WEINSTEIN J A,RAN F A,KONERMANNS,AGARWALA V,LI Y,FINE E J,WU X,SHALEM O,CRADICKT J,MARRAFFINI L A,BAO G,ZHANG F.DNA targeting specificity of RNA-guided Cas9 nucleases.Nature Biotechnology,2013,31(9):827-832.
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[43]RAN F A,CONG L,YAN W X,SCOTT D A,GOOTENBERG J S,KRIZ A J,ZETSCHE B,SHALEM O,WU X,MAKAROVA K S,KOONIN E V,SHARP P A,ZHANG F.In vivo genome editing using Staphylococcus aureus Cas9.Nature,2015,520(7546):186-191.
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