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Systematic prediction of genes functionally linked to CRISPR-Cas systems by gene neighborhood analysis.

ABSTRACT: The CRISPR-Cas systems of bacterial and archaeal adaptive immunity consist of direct repeat arrays separated by unique spacers and multiple CRISPR-associated (cas) genes encoding proteins that mediate all stages of the CRISPR response. In addition to the relatively small set of core cas genes that are typically present in all CRISPR-Cas systems of a given (sub)type 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, but the functional relevance of most remains unknown. We developed a computational strategy for systematically detecting genes that are likely to be functionally linked to CRISPR-Cas. 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 cas genes are considered candidate CRISPR-linked genes. We describe additional criteria to predict functionally relevance for genes in the candidate set and identify 79 genes as strong candidates for functional association with CRISPR-Cas systems. A substantial majority of these CRISPR-linked genes reside in type III CRISPR-cas loci, which implies exceptional functional versatility of type III systems. Numerous candidate CRISPR-linked genes encode integral membrane proteins suggestive of tight membrane association of CRISPR-Cas systems, whereas many others encode 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 system functionality.

SUBMITTER: Shmakov SA

PROVIDER: S-EPMC6003329 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Systematic prediction of genes functionally linked to CRISPR-Cas systems by gene neighborhood analysis.

Shmakov Sergey A SA Makarova Kira S KS Wolf Yuri I YI Severinov Konstantin V KV Koonin Eugene V EV

Proceedings of the National Academy of Sciences of the United States of America 20180521 23

The CRISPR-Cas systems of bacterial and archaeal adaptive immunity consist of direct repeat arrays separated by unique spacers and multiple CRISPR-associated (cas) genes encoding proteins that mediate all stages of the CRISPR response. In addition to the relatively small set of core cas genes that are typically present in all CRISPR-Cas systems of a given (sub)type and are essential for the defense function, numerous genes occur in CRISPR-cas loci only sporadically. Some of ...[more]

PMID: 29784811

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Project description:Novel CRISPR-Cas systems possess substantial potential for genome editing and manipulation of gene expression. The types and numbers of CRISPR-Cas systems vary substantially between different organisms. Some filamentous cyanobacteria harbor > 40 different putative CRISPR repeat-spacer cassettes, while the number of cas gene instances is much lower. Here we addressed the types and diversity of CRISPR-Cas systems and of CRISPR-like repeat-spacer arrays in 171 publicly available genomes of multicellular cyanobacteria. The number of 1328 repeat-spacer arrays exceeded the total of 391 encoded Cas1 proteins suggesting a tendency for fragmentation or the involvement of alternative adaptation factors. The model cyanobacterium Anabaena sp. PCC 7120 contains only three cas1 genes but hosts three Class 1, possibly one Class 2 and five orphan repeat-spacer arrays, all of which exhibit crRNA-typical expression patterns suggesting active transcription, maturation and incorporation into CRISPR complexes. The CRISPR-Cas system within the element interrupting the Anabaena sp. PCC 7120 fdxN gene, as well as analogous arrangements in other strains, occupy the genetic elements that become excised during the differentiation-related programmed site-specific recombination. This fact indicates the propensity of these elements for the integration of CRISPR-cas systems and points to a previously not recognized connection. The gene all3613 resembling a possible Class 2 effector protein is linked to a short repeat-spacer array and a single tRNA gene, similar to its homologs in other cyanobacteria. The diversity and presence of numerous CRISPR-Cas systems in DNA elements that are programmed for homologous recombination make filamentous cyanobacteria a prolific resource for their study. Abbreviations: Cas: CRISPR associated sequences; CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats; C2c: Class 2 candidate; SDR: small dispersed repeat; TSS: transcriptional start site; UTR: untranslated region.

Project description:CRISPR-Cas systems are prokaryotic adaptive immune systems that facilitate protection of bacteria and archaea against infection by external mobile genetic elements. The model pathogen Francisella novicida encodes a CRISPR-Cas12a (FnoCas12a) system and a CRISPR-Cas9 (FnoCas9) system, the latter of which has an additional and noncanonical function in bacterial virulence. Here, we investigated and compared the functional roles of the FnoCas12a and FnoCas9 systems in transformation inhibition and bacterial virulence. Unlike FnoCas9, FnoCas12a was not required for F. novicida virulence. However, both systems were highly effective at plasmid restriction and acted independently of each other. We further identified a critical protospacer-adjacent motif (PAM) necessary for transformation inhibition by FnoCas12a, demonstrating a greater flexibility for target identification by FnoCas12a than previously appreciated and a specificity that is distinct from that of FnoCas9. The effectors of the two systems exhibited different patterns of expression at the mRNA level, suggesting that they may confer distinct benefits to the bacterium in diverse environments. These data suggest that due to the differences between the two CRISPR-Cas systems, together they may provide F. novicida with a more comprehensive defense against foreign nucleic acids. Finally, we demonstrated that the FnoCas12a and FnoCas9 machineries could be simultaneously engineered to restrict the same nonnative target, thereby expanding the toolset for prokaryotic genome manipulation.IMPORTANCE CRISPR-Cas9 and CRISPR-Cas12a systems have been widely commandeered for genome engineering. However, they originate in prokaryotes, where they function as adaptive immune systems. The details of this activity and relationship between these systems within native host organisms have been minimally explored. The human pathogen Francisella novicida contains both of these systems, with the Cas9 system also exhibiting a second activity, modulating virulence through transcriptional regulation. We compared and contrasted the ability of these two systems to control virulence and restrict DNA within their native host bacterium, highlighting differences and similarities in these two functions. Collectively, our results indicate that these two distinct and reprogrammable endogenous systems provide F. novicida with a more comprehensive defense against mobile genetic elements.

Dataset Information

Systematic prediction of genes functionally linked to CRISPR-Cas systems by gene neighborhood analysis.

Publications

Systematic prediction of genes functionally linked to CRISPR-Cas systems by gene neighborhood analysis.

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