A Guild of 45 CRISPR-Associated (Cas) Protein Families and Multiple CRISPR/Cas Subtypes Exist in Prokaryotic Genomes

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPRs) are a family of DNA direct repeats found in many prokaryotic genomes. Repeats of 21–37 bp typically show weak dyad symmetry and are separated by regularly sized, nonrepetitive spacer sequences. Four CRISPR-associated (Cas) protein families, designated Cas1 to Cas4, are strictly associated with CRISPR elements and always occur near a repeat cluster. Some spacers originate from mobile genetic elements and are thought to confer “immunity” against the elements that harbor these sequences. In the present study, we have systematically investigated uncharacterized proteins encoded in the vicinity of these CRISPRs and found many additional protein families that are strictly associated with CRISPR loci across multiple prokaryotic species. Multiple sequence alignments and hidden Markov models have been built for 45 Cas protein families. These models identify family members with high sensitivity and selectivity and classify key regulators of development, DevR and DevS, in Myxococcus xanthus as Cas proteins. These identifications show that CRISPR/cas gene regions can be quite large, with up to 20 different, tandem-arranged cas genes next to a repeat cluster or filling the region between two repeat clusters. Distinctive subsets of the collection of Cas proteins recur in phylogenetically distant species and correlate with characteristic repeat periodicity. The analyses presented here support initial proposals of mobility of these units, along with the likelihood that loci of different subtypes interact with one another as well as with host cell defensive, replicative, and regulatory systems. It is evident from this analysis that CRISPR/cas loci are larger, more complex, and more heterogeneous than previously appreciated.

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

“…Archaeal specific genes (csa1-csa5) reported to show a degree of specificity across archaeal or bacterial phyla (Bolotin et al 2005;Haft et al 2005;Cooper and Overstreet 2014). Therefore, the cas genes of most thermophilic genomes have appeared in the order of cas5-cas3-cas1-cas2.…”

“…Clustered regularly interspaced short palindromic repeats (CRISPRs) have played an important role in the chromosomal rearrangement, DNA mobilization, repair and regulation and also replicon partitioning (Haft et al 2005;DeBoy et al 2006;Makarova et al 2006;Cooper and Overstreet 2014). Among 25 gene families in the CAS system, eight gene families (cas1, cas2, cas4, cas6, cas7) have reported for the nuclease activity, nine families (cas5) for Repeat-Associated Mysterious Proteins (RAMP) and two families (cas3) for DNA/RNA polymerase/helicase activity (Makarova et al 2006;Quax et al 2013;Vestergaard et al 2014).…”

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

“…• Processes the CRISPR precursor crRNAs to release invaderspecific crRNAs [16,37] Cas genes that are universal to all CRISPR-Cas systems, although Cas3 through Cas6 are also very widespread [63,122]. The core Cas genes are known to be adjacent to the CRISPR locus, and a summary of their known characteristics and functions can be found in Table 1.…”

“…Cse3 and Cas6 do however contain similar structures and RNA-recognition motifs, even though they lack sequence homology [16,63,126]. In bacteria containing Csn subtype (Type II) CRISPR systems, an RNA transcribed from nearby to the CRISPR locus anneals to the repeat sequence of the pre-crRNA.…”

“…Comparisons of Cas gene sequences, adaptation mechanisms, and crRNA processing mechanisms have demonstrated that even though Bacteria and Archaea represent two entirely different domains of life, and Archaea is more closely related to eukaryotes than bacteria, the CRISPR-Cas systems found in these two domains are indeed very similar [63,64,98].…”