Analysis of CRISPR-Cas System in Streptococcus thermophilus and Its Application

Abstract

CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR associated proteins) loci, which provide a specific immunity against exogenous elements, are hypervariable among distinct prokaryotes. Based on previous researches, this review focuses on concluding systematical genome editing protocols in Streptococcus thermophilus. Firstly, its protocols and optimized conditions in gene editing are introduced. What's more, classification and diversity analyses of S. thermophilus CRISPR-Cas benefit the further understanding of evolution relationship among Streptococcus. Ability of its foreign segment integration and spacer source analyses also indicate a new direction of phage resistance. Above all, all of these point out its potential to be regarded as another model system other than type II CRISPR-Cas in Streptococcus pyogenes.

Keywords: CRISPR-Cas; Streptococcus thermophilus; diversity; gene editing; model system.

FIGURE 1

Diagram of CRISPR/Cas9 system-mediated gene editing process. pCas9 plasmid consists of cas9 gene, sgRNA, resistance locus and homologous arms. Cas9 and sgRNA gene express in order to identify target DNA and cleave at specific sites in front of PAM. RuvC and HNH are both nuclease domain in Cas9, and they cleave target DNA strand and its antisense strand, respectively. Homologous arm sequences in plasmid are complementary to their counterparts in genome so that the impact target gene fragment can be selected accurately. The whole target DNA is able to be deleted, replaced or added with a piece of new sequence through design of different homologous repair arm.

FIGURE 2

Structure of S. thermophilus CRISPR3 and S. pyogenes type II CRISPR-Cas loci and their homology comparison. The spacers and repeat sequences are represented by colored rhombi and black columns, respectively. The homology percentage between four Cas protein genes and repeat sequences are indicated in the gray shading. (S. thermophilus ASCC 1275 NCBI Reference Sequence: NZ_CP006819.1 and S. pyogenes NCBI Reference Sequence: NC_007296.1).