Diversity, Activity, and Evolution of CRISPR Loci in Streptococcus thermophilus

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) are hypervariable loci widely distributed in prokaryotes that provide acquired immunity against foreign genetic elements. Here, we characterize a novel Streptococcus thermophilus locus, CRISPR3, and experimentally demonstrate its ability to integrate novel spacers in response to bacteriophage. Also, we analyze CRISPR diversity and activity across three distinct CRISPR loci in several S. thermophilus strains. We show that both CRISPR repeats and cas genes are locus specific and functionally coupled. A total of 124 strains were studied, and 109 unique spacer arrangements were observed across the three CRISPR loci. Overall, 3,626 spacers were analyzed, including 2,829 for CRISPR1 (782 unique), 173 for CRISPR2 (16 unique), and 624 for CRISPR3 (154 unique). Sequence analysis of the spacers revealed homology and identity to phage sequences (77%), plasmid sequences (16%), and S. thermophilus chromosomal sequences (7%). Polymorphisms were observed for the CRISPR repeats, CRISPR spacers, cas genes, CRISPR motif, locus architecture, and specific sequence content. Interestingly, CRISPR loci evolved both via polarized addition of novel spacers after exposure to foreign genetic elements and via internal deletion of spacers. We hypothesize that the level of diversity is correlated with relative CRISPR activity and propose that the activity is highest for CRISPR1, followed by CRISPR3, while CRISPR2 may be degenerate. Globally, the dynamic nature of CRISPR loci might prove valuable for typing and comparative analyses of strains and microbial populations. Also, CRISPRs provide critical insights into the relationships between prokaryotes and their environments, notably the coevolution of host and viral genomes.

Knowledge-based discovery for designing CRISPR-CAS systems against invading mobilomes in thermophiles

“…Section A is representing a genomic organization of CAS gene clusters and associated CRISPR loci whereas section B is showing the proposed molecular mechanism of xsiRNA processing to cleave the target RNAs. xsiRNA: extremophilic short interfering RNA; xRISC: extremophilic RNA induced silencing complexes their invading phages and plasmids (Horvath et al 2008). Also, the prophages account on their genetic variation across closely related strains and contribute in evolution of adaptive immune systems, which was agreed to earlier works (Marraffini and Sontheimer 2009;Bondy-Denomy and Davidson 2014).…”

“…Non-repetitive spacer sequences have suggested as a potential target site for the genome invaders in many archaeal genomes (Brügger et al 2002). CRISPR loci have shown a distinct genetic and molecular diversity in the strains of a thermophilic archaean, Methanocaldococcus jannaschii (Bult et al 1996) and of thermophilic bacterium, Streptococcus thermophilus (Horvath et al 2008). Some of the CRISPR sequences in Thermoanaerobacter tengcongensis and Thermus thermophilus HB8 chromosomes have shared with their existing megaplasmids (Jansen et al 2002;Godde and Bickerton 2006).…”

Strong bias in the bacterial CRISPR elements that confer immunity to phage

“…There are 233 candidate proto-spacers that can be theoretically acquired from D2972 into CRISPR1 (AGAAW PAM, frequency of 95.19%), and 483 into CRISPR3 (GGNG PAM, frequency of 96.68%) 19 . In both experiments, we noted that some specific CRISPR1 and CRISPR3 spacers were highly represented in the set of newly acquired spacers.…”

Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems

“…The CRISPR locus is rarely targeted by integrative genetic elements, even in species in which genetic shuffling caused by these mobile sequences is common. Nonetheless, transpositions do on occasion occur, and interrupt the usual spacer-repeat sequence of the CRISPR locus [74,119].…”

“…This horizontal transfer of CRISPR loci has been documented in many cases. For example, transfer of the CRISPR locus and Cas genes has been implicated between lactic acid bacteria and crenarchaea [74,91,119].…”