Comprehensive search for accessory proteins encoded with archaeal and bacterial type III CRISPR-cas gene cassettes reveals 39 new cas gene families

Abstract

A study was undertaken to identify conserved proteins that are encoded adjacent to cas gene cassettes of Type III CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR associated) interference modules. Type III modules have been shown to target and degrade dsDNA, ssDNA and ssRNA and are frequently intertwined with cofunctional accessory genes, including genes encoding CRISPR-associated Rossman Fold (CARF) domains. Using a comparative genomics approach, and defining a Type III association score accounting for coevolution and specificity of flanking genes, we identified and classified 39 new Type III associated gene families. Most archaeal and bacterial Type III modules were seen to be flanked by several accessory genes, around half of which did not encode CARF domains and remain of unknown function. Northern blotting and interference assays in Synechocystis confirmed that one particular non-CARF accessory protein family was involved in crRNA maturation. Non-CARF accessory genes were generally diverse, encoding nuclease, helicase, protease, ATPase, transporter and transmembrane domains with some encoding no known domains. We infer that additional families of non-CARF accessory proteins remain to be found. The method employed is scalable for potential application to metagenomic data once automated pipelines for annotation of CRISPR-Cas systems have been developed. All accessory genes found in this study are presented online in a readily accessible and searchable format for researchers to audit their model organism of choice: http://accessory.crispr.dk .

Type IV CRISPR-Cas systems are highly diverse and involved in competition between plasmids

“…Following manual annotation, the protein sequences from the refined representative modules were pooled for another all-against-all sequence comparison. Protein sequences were clustered using the method previously described 72 and another aggregate module similarity tree was built for the refined representative type IV module set. The tree was overlaid with gene maps of the type IV modules marked with the obtained protein clustering information ( Supplementary Fig.…”

“…Enrichment in spacer targeting of certain functions was done by first predicting ORFs in all plasmid and phage genomes using Prodigal 78 , and then clustering genes using the protein clustering algorithm previously described 72 . The observed number of matches to each gene cluster was compared to 10 5 simulations of random draws from a binomial distribution with size n equal to the number of genes in the gene cluster and the probability = # %& '()*'+ ,-./0)(1 23-/+42 # %& 1+(+2 * ℎ, where the relative gene length was defined by dividing the length of each gene by the median gene length, and then finding the average length for each gene cluster.…”

CasLocusAnno: a web‐based server for annotating cas loci and their corresponding (sub)types

“…identified some co‐functional accessory genes within the type III CRISPR‐Cas system using a guilt‐by‐association approach and Makarova et al . 's cas loci data . Self‐targeting (ST) occurs when a spacer and its targeting segment appear in the same prokaryotic organism, leading to reduced bacterial fitness.…”

“…Up to now, some specific Cas proteins have been discovered such as Cas9 targeting dsDNA, Cpf1 targeting dsDNA, C2c2 targeting ssRNA [2] and Cas14 targeting ssDNA [30]. [1,33]. Self-targeting (ST) occurs when a spacer and its targeting segment appear in the same prokaryotic organism, leading to reduced bacterial fitness.…”

CRISPRCasIdentifier: Machine learning for accurate identification and classification of CRISPR-Cas systems

“…Type III systems, however, use the Cas7 backbone for cleaving invader mRNA while the Cas10 HD nuclease cleaves transcribed DNA (7,8). Cas10 also synthesises a signaling molecule that recruits additional accessory Cas proteins for other functions, such as cell suicide or activation of other defense systems (8,9).…”