Severinov, K. V. | Search bioRxiv

bioRxiv2018

Towards comprehensive characterization of CRISPR-linked genes

The CRISPR-Cas systems of bacterial and archaeal adaptive immunity consist of arrays of direct repeats separated by unique spacers and multiple CRISPR-associated (cas) genes encoding proteins that mediate the adaptation, CRISPR RNA maturation and interference stages of the CRISPR response. In addition to the relatively small set of core cas genes that are typically present in all representatives of each (sub)type of CRISPR-Cas systems and are essential for the defense function, numerous genes occur in CRISPR-cas loci only sporadically. Some of these have been shown to perform various ancillary roles in CRISPR response whereas the functional relevance of many others, if any, remains obscure. We developed a computational strategy for systematically detecting genes that are likely to be functionally linked to CRISPR-Cas systems. The approach is based on a \"CRISPRicity\" metric that measures the strength of CRISPR association for all protein-coding genes from sequenced bacterial and archaeal genomes. Uncharacterized genes with CRISPRicity values comparable to those of known cas genes are considered candidate CRISPR-ancillary genes, and we describe additional criteria to identify functionally relevant genes in the candidate set. About 80 genes that were not previously reported to be associated with CRISPR-Cas were identified as probable CRISPR-ancillary genes. A substantial majority of these genes reside in type III CRISPR-cas loci which implies exceptional functional versatility of type III systems. Numerous candidate CRISPR-ancillary genes encode integral membrane proteins suggestive of tight membrane connections of type III CRISPR-Cas whereas many other candidates are proteins implicated in various signal transduction pathways. These predictions provide ample material for improving annotation of CRISPR-cas loci and experimental characterization of previously unsuspected aspects of CRISPR-Cas functionality.\n\nSIGNIFICANCEThe CRISPR-Cas systems that mediate adaptive immunity in bacteria and archaea encompass a small set of core cas genes that are essential in a broad range of CRISPR-Cas systems. However, a much greater number of genes only sporadically co-occur with CRISPR-Cas, and for most of these, involvement in CRISPR-Cas functions has not been demonstrated. We developed a computational strategy that provides for systematic identification of CRISPR-linked proteins and prediction of their functional association with CRISPR-Cas systems. About 80 previously undetected, putative CRISPR-accessory proteins were identified. A large fraction of these proteins are predicted to be membrane-associated revealing an unknown side of CRISPR biology.

genomics↗

bioRxiv2017

The CRISPR Spacer Space Is Dominated By Sequences From The Species-Specific Mobilome

The CRISPR-Cas is the prokaryotic adaptive immunity system that stores memory of past encounters with foreign DNA in spacers that are inserted between direct repeats in CRISPR arrays 1,2. Only for a small fraction of the spacers, homologous sequences, termed protospacers, are detectable in viral, plasmid or microbial genomes 3,4. The rest of the spacers remain the CRISPR \"dark matter\". We performed a comprehensive analysis of the spacers from all CRISPR-cas loci identified in bacterial and archaeal genomes, and found that, depending on the CRISPR-Cas subtype and the prokaryotic phylum, protospacers were detectable for 1 to about 19% of the spacers ([~]7% global average). Among the detected protospacers, the majority, typically, 80 to 90%, originate from viral genomes, and among the rest, the most common source are genes integrated in microbial chromosomes but involved in plasmid conjugation or replication. Thus, almost all spacers with identifiable protospacers target mobile genetic elements (MGE). The GC-content, as well as dinucleotide and tetranucleotide compositions, of microbial genomes, their spacer complements, and the cognate viral genomes show a nearly perfect correlation and are almost identical. Given the near absence of self-targeting spacers, these findings are best compatible with the possibility that the spacers, including the dark matter, are derived almost completely from the species-specific microbial mobilomes.

microbiology↗