干酪乳杆菌CRISPR基因座分析

doi:10.3864/j.issn.0578-1752.2019.03.012

Abstract

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

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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Figures/Tables 7

Fig. 1

Table 1

Fig. 2

Fig. 3

Fig. 4

Table 2

Sequence characteristics of the original spacer corresponding to L. casei spacer"

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

Table 2

Fig. 5

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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

CRISPR Locus Analysis of Lactobacillus casei

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