Department of Food Science and Engineering, School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin, 150001, People's Republic of China.
Institute of Microbiology, Heilongjiang Academy of Sciences, Harbin, 150010, People's Republic of China.
Arch Microbiol. 2020 May;202(4):695-710. doi: 10.1007/s00203-019-01780-3. Epub 2019 Nov 28.
Clustered regularly interspaced short palindromic repeats (CRISPR) consists of a series of regular repeat-spacer sequences. It can not only act as a natural immune system in most prokaryotes, but also be utilized as the tool of newly developed genome modification and evolutionary researches. Streptococcus thermophilus is an important model organism for the study and application of CRISPR systems. In present study, the occurrence and diversity of CRISPR-Cas systems in the genomes of S. thermophilus were investigated including 4 new sequenced strains CS5, CS9, CS18, CS20, and other 23 strains downloaded from NCBI website. 66 CRISPR/Cas systems were identified among these 27 strains and could divided into four subsystems according to the arrangement of Cas proteins, notably I-E, II-A, II-C and III-A. Overall, 26 type II-C systems, 18 type II-A systems, 13 type III-A systems, 9 type I-E systems were identified. It was mentioned that CS20 contained two type II-C systems which had not been identified in the other 26 S. thermophilus strains. Overall, 1,080 spacers were analyzed and blasted. Sequence identity searches of spacers implied that most spacers derived from partial sequences of exogenous DNA, including various bacteriophages and plasmids. Of note, a large number of novel spacers were found in this study, indicating the unique phage environment they have undergone, especially CS20 strain. In addition, the analysis of the cas1 and cas9 genes revealed the genetic relationship among CRISPR-Cas system in these strains. Furthermore, the analysis of CRISPR spacers also indicated protospacer adjacent motif (PAM) sequences. Summary of PAM sequences could lay the foundations for the application of S. thermophilus CRISPR-Cas system. Our results suggested CS5 and CS18 can be used as model strains in the research of CRISPR-Cas system, and CS20 might have greater application potential in gene editing.
簇状规律间隔短回文重复序列（CRISPR）由一系列规则重复间隔序列组成。它不仅可以作为大多数原核生物的天然免疫系统，还可以作为新开发的基因组修饰和进化研究的工具。嗜热链球菌是研究和应用 CRISPR 系统的重要模式生物。本研究调查了嗜热链球菌基因组中 CRISPR-Cas 系统的发生和多样性，包括新测序的 4 株 CS5、CS9、CS18 和 CS20 以及从 NCBI 网站下载的其他 23 株菌株。在这 27 株菌中鉴定出 66 个 CRISPR/Cas 系统，根据 Cas 蛋白的排列可分为四个亚系统，分别为 I-E、II-A、II-C 和 III-A。总体而言，鉴定出 26 个 II-C 型系统、18 个 II-A 型系统、13 个 III-A 型系统和 9 个 I-E 型系统。值得注意的是，CS20 含有两个尚未在其他 26 株嗜热链球菌中鉴定出的 II-C 型系统。总体上分析了 1080 个间隔区并进行了比对。间隔区的序列同一性搜索表明，大多数间隔区来源于外源 DNA 的部分序列，包括各种噬菌体和质粒。值得注意的是，本研究发现了大量新的间隔区，表明它们经历了独特的噬菌体环境，尤其是 CS20 菌株。此外，对 cas1 和 cas9 基因的分析揭示了这些菌株中 CRISPR-Cas 系统的遗传关系。此外，对 CRISPR 间隔区的分析也表明了前导相邻基序（PAM）序列。PAM 序列的总结为嗜热链球菌 CRISPR-Cas 系统的应用奠定了基础。我们的研究结果表明 CS5 和 CS18 可作为 CRISPR-Cas 系统研究的模型菌株，CS20 在基因编辑方面可能具有更大的应用潜力。
