Characterization and comparison of CRISPR Loci in Streptococcus thermophilus

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.