中国农业科学 ›› 2019, Vol. 52 ›› Issue (3): 521-529.doi: 10.3864/j.issn.0578-1752.2019.03.012
杨兰,杨洋(),李伟勋,ObaroakpoJOY,逄晓阳(
),吕加平(
)
收稿日期:
2018-08-03
接受日期:
2018-11-14
出版日期:
2019-02-01
发布日期:
2019-02-14
作者简介:
杨兰,E-mail: 基金资助:
YANG Lan,YANG Yang(),LI WeiXun,OBAROAKPO JOY,PANG XiaoYang(
),LÜ JiaPing(
)
Received:
2018-08-03
Accepted:
2018-11-14
Online:
2019-02-01
Published:
2019-02-14
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。
杨兰,杨洋,李伟勋,ObaroakpoJOY,逄晓阳,吕加平. 干酪乳杆菌CRISPR基因座分析[J]. 中国农业科学, 2019, 52(3): 521-529.
YANG Lan,YANG Yang,LI WeiXun,OBAROAKPO JOY,PANG XiaoYang,LÜ JiaPing. CRISPR Locus Analysis of Lactobacillus casei[J]. Scientia Agricultura Sinica, 2019, 52(3): 521-529.
表1
6株干酪乳杆菌CRISPR序列情况"
菌株 Strain | CRISPR长度 Length of CRISPR | Spacer数量 Number of spacer | DR序列长度 Length of DR sequence | DR序列 DR sequence | Cas |
---|---|---|---|---|---|
L. casei ZHANG | 1092 | 16 | 36 | GTCTCAGGTAGATGTCGAATCAATCAGTTCAAGAGC | Cas9, Cas1, Cas2, Csn2 |
L. casei ZHANG | 145 | 1 | 46 | GGGGTCCTTATGAGCAGGTTTCTGCGCCTGTTTGCGCGTTTCGAAA | 无 No |
L. casei ZHANG | 109 | 1 | 27 | GGTCCTTACACGTAGGTTTCTGGTCTG | 无 No |
L. casei ZHANG | 116 | 1 | 31 | CTTTGGTCGTTTAGGTTCGAGGTCCTTATGC | 无 No |
L. casei ZHANG | 121 | 1 | 33 | CGGTTTCTAAACGCGTTCGCCACCCCAGAAACC | 无 No |
L. casei ZHANG | 107 | 1 | 29 | GGTCCTTATGTGTAGGTTTCTGGGCCAGC | 无 No |
L. casei ZHANG 质粒 plca36 | 78 | 1 | 24 | AAAGTCCGCATGACTTCGTTGAAA | 无 No |
L. casei BD-Ⅱ | 1422 | 21 | 36 | GTCTCAGGTAGATGTCGAATCAATCAGTTCAAGAGC | Cas9, Cas1, Cas2, Csn2 |
L. casei BL23 | 1422 | 21 | 36 | GTCTCAGGTAGATGTCGAATCAATCAGTTCAAGAGC | Cas9, Cas1, Cas2, Csn2 |
L. casei LC2W | 1422 | 21 | 36 | GTCTCAGGTAGATGTCGAATCAATCAGTTCAAGAGC | Cas9, Cas1, Cas2, Csn2 |
L. casei W56 | 1424 | 21 | 36 | GTCTCAGGTAGATGTCGAATCAATCAGTTCAAGAGC | Cas9, Cas1, Cas2, Csn2 |
L. casei LOCK919 | 762 | 11 | 36 | GTCTCAGGTAGATGTCGAATCAATCAGTTCAAGAGC | Cas9, Cas1, Cas2, Csn2 |
L. casei LOCK919 质粒 pLOCK919 | 146 | 2 | 26 | CGGGAAACCGAAAATCGGTCGCCCGC | 无 No |
表2
干酪乳杆菌spacer对应的原间隔区序列特点"
间隔 Spacer | 原间隔区 Original interval | 开放阅读框 Open reading box | 匹配性 Matching |
---|---|---|---|
BD-Ⅱ间隔区21 BD-Ⅱspacer21 | 乳杆菌质粒pREN Lactobacillus rennini plasmid pREN | 假定蛋白质 Assuming protein | 30/30 |
BL23间隔区21 BL23 spacer21 | 乳杆菌质粒pREN Lactobacillus rennini plasmid pREN | 假定蛋白质 Assuming protein | 30/30 |
LC2W 间隔区21 LC2W spacer21 | 乳杆菌质粒pREN Lactobacillus rennini plasmid pREN | 假定蛋白质 Assuming protein | 30/30 |
W56 间隔区21 W56 spacer21 | 乳杆菌质粒pREN Lactobacillus rennini plasmid pREN | 假定蛋白质 Assuming protein | 30/30 |
ZHANG间隔区14 ZHANG spacer14 | 乳酸乳球菌UL8质粒pUL8C Lactococcus lactis subsp. lactis strain UL8 plasmid pUL8C | ─ | 29/30 |
乳酸乳球菌乳脂亚种UC109 质粒pUC109F Lactococcus lactis subsp. cremoris strain UC109 plasmid pUC109F | ─ | 29/30 | |
乳酸乳球菌C10 质粒pC10A Lactococcus lactis subsp. lactis strain C10 plasmid pC10A | ─ | 29/30 | |
乳酸乳球菌KLDS 4.0325 质粒 unnamed2 Lactococcus lactis subsp. lactis KLDS 4.0325 plasmid unnamed2 | ─ | 29/30 | |
乳酸乳球菌质粒pCL2.1 Lactococcus lactis plasmid pCL2.1 | ─ | 29/30 | |
ZHANG间隔区15 ZHANG spacer15 | 乳酸杆菌TMW 1.1992 质粒 pL11992-8 Lactobacillus backii strain TMW 1.1992 plasmid pL11992-8 | ─ | 29/30 |
副干酪乳杆菌质粒pLP5403 Lactobacillus paracasei plasmid pLP5403 | 假定蛋白质 Assuming protein | 29/30 | |
卡氏双球菌ATCC BAA-344 质粒 pPECL-1 Pediococcus claussenii ATCC BAA-344 plasmid pPECL-1 | ─ | 29/30 | |
植物乳杆菌质粒pXY3 Lactobacillus plantarum plasmid pXY3 | ORF4 | 29/30 | |
短乳酸杆菌质粒pLB925A01 Lactobacillus brevis plasmid pLB925A01 | ─ | 29/30 | |
乳明串珠球菌质粒pCI411 Leuconostoc lactis plasmid pCI411 | ─ | 29/30 | |
植物乳杆菌MF1298质粒19 Lactobacillus plantarum strain MF1298 plasmid unnamed19 | ─ | 28/30 | |
戊糖片球菌SRCM100892质粒pPC892-5 Pediococcus pentosaceus strain SRCM100892 plasmid pPC892-5 | ─ | 27/30 |
[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.
doi: 10.1021/sb500351f pmid: 25458909 |
[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.
doi: 10.1128/AEM.01248-15 pmid: 26002895 |
[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, ZHAO J . 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 H J . Probiotic beverage containing Lactobacillus casei Shirota improves gastrointestinal symptoms in patients with chronic constipation. Canadian Journal of Gastroenterology, 2016,17(11):655-659.
doi: 10.1155/2003/654907 pmid: 14631461 |
[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.
doi: 10.1016/j.jff.2015.05.021 |
[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.
doi: 10.1093/nar/gkt157 pmid: 23470997 |
[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, KROOS L . 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.
doi: 10.1128/JB.00187-07 |
[10] | KARVELIS T, GASIUNAS G, MIKSYS A, BARRANGOU R, HORVATH P, SIKSNYS V . CrRNA and tracrRNA guide Cas9- mediated DNA interference in Streptococcus thermophilus. RNA Biology, 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, CHARPENTIER E . CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature, 2011,471(7340):602-607.
doi: 10.1038/nature09886 pmid: 21455174 |
[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.
doi: 10.1016/j.tibs.2009.05.002 pmid: 19646880 |
[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.
doi: 10.1016/j.bbrc.2014.01.141 pmid: 24491566 |
[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.
doi: 10.1186/s13059-015-0653-x pmid: 25924609 |
[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 freeze- thawed fertilized oocytes. BMC Biotechnology, 2015,15(1):33.
doi: 10.1186/s12896-015-0144-x pmid: 25997509 |
[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.
doi: 10.1038/cr.2013.157 pmid: 24296780 |
[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.
doi: 10.1101/gr.186379.114 pmid: 26048245 |
[18] |
AUER T O, DUROURE K, DE CIAN A, CONCORDET J P, DEL BENE F . Highly efficient CRISPR/Cas9 mediated knock-in in zebrafish by homology-independent DNA repair. Genome Research, 2014,24(1):142.
doi: 10.1101/gr.161638.113 pmid: 24179142 |
[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.
doi: 10.1038/cr.2013.123 |
[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.
doi: 10.1021/sb500036q pmid: 4277763 |
[24] |
JINEK M, CHYLINSKI K, FONFARA I, HAUER M, DOUDNA J A, CHARPENTIER E . A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012,337(6096):816-821.
pmid: 22745249 |
[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.
doi: 10.1038/nbt.2508 pmid: 23360965 |
[26] |
BURGESS D J . Technology: A CRISPR genome-editing tool. Nature Reviews Genetics, 2013,14(2):80.
doi: 10.1038/nrg3409 pmid: 23322222 |
[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.
doi: 10.1111/mmi.12678 pmid: 24942885 |
[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.
doi: 10.4161/rna.24321 pmid: 23563642 |
[30] |
HORVATH P, BARRANGOU R . CRISPR/Cas, the immune system of bacteria and archaea. Science, 2010,327(5962):167-170.
doi: 10.1126/science.1179555 pmid: 20056882 |
[31] |
BARRANGOU R, FREMAUX C, DEVEAU H, RICHARDS M, BOYAVAL P, MOINEAU S, ROMERO D A, HORVATH P . CRISPR provides acquired resistance against viruses in prokaryotes. Science, 2007,315(5819):1709-1712.
doi: 10.1126/science.1138140 pmid: 17379808 |
[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.
doi: 10.1126/science.1165771 pmid: 2695655 |
[35] |
BARRANGOU R, HORVATH P . A decade of discovery: CRISPR functions and applications. Nature Microbiology, 2017,2:17092.
doi: 10.1038/nmicrobiol.2017.92 pmid: 28581505 |
[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.
doi: 10.1371/journal.pone.0133661 pmid: 4521832 |
[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, UEDA K, STAHL B, BARRANGOU R, BANFIELD J F . Strong bias in the bacterial CRISPR elements that confer immunity to phage. Nature Communications, 2013,4:1430.
doi: 10.1038/ncomms2440 pmid: 23385575 |
[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.
doi: 10.1073/pnas.1208507109 |
[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.
doi: 10.1099/mic.0.023960-0 pmid: 19246744 |
[41] |
HSU P D, SCOTT D A, WEINSTEIN J A, RAN F A, KONERMANN S, AGARWALA V, LI Y, FINE E J, WU X, SHALEM O, CRADICK T J, MARRAFFINI L A, BAO G, ZHANG F . DNA targeting specificity of RNA-guided Cas9 nucleases. Nature Biotechnology, 2013,31(9):827-832.
doi: 10.1038/nbt.2647 |
[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.
doi: 10.1038/srep05405 pmid: 4066725 |
[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 PAM recognition. Nature Biotechnology, 2015,33(12):1293-1298. |
[1] | 李红娟, 刘鹭, 张书文, 孔凡丕, 孙卓, 吕加平. Lactobacillus casei AST18抗真菌特性及其在酸奶保鲜中的应用[J]. 中国农业科学, 2011, 44(19): 4050-4057. |
[2] | 张书文,吕加平,孟和毕力格,刘鹭,胡鲜宝 . 干酪乳杆菌干酪亚种Lactobacillus casei subsp.casei SY13对衰老模型小鼠的抗氧化作用 [J]. 中国农业科学, 2010, 43(10): 2141-2146 . |
[3] | 吴延博,陈从英,张志燕,郭源梅,高 军. 猪精子黏合分子1(SPAM1)基因在白色杜洛克×二花脸F2资源群体中的遗传变异及其与母猪产仔数的关联性[J]. 中国农业科学, 2009, 42(6): 2111-2117 . |
[4] | 徐义刚,崔丽春,葛俊伟,唐丽杰,赵丽丽,李一经. 表达猪细小病毒VP2蛋白的重组干酪乳杆菌诱导小鼠产生特异性抗体[J]. 中国农业科学, 2008, 41(3): 846-851 . |
[5] | 许梓荣,陈洪亮,肖日进. Ractopamine对肥育猪营养再分配作用机制的研究[J]. 中国农业科学, 1998, 31(06): 69-75 . |
|