干酪乳杆菌CRISPR基因座分析

doi:10.3864/j.issn.0578-1752.2019.03.012

摘要/Abstract

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。

关键词: 干酪乳杆菌, CRISPR系统, spacer, Cas, PAM

Abstract:

【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 1 ^st and 3 ^rd bases. The 2 ^nd and 4 ^th 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.

Key words: Lactobacillus casei, CRISPR system, spacer, Cas gene, PAM

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.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2019.03.012

https://www.chinaagrisci.com/CN/Y2019/V52/I3/521

图/表 7

图1

表1

图2

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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

表2

图5

参考文献 44

[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.

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