Biology subjects

Makarova, K. S.

Publications and source records attributed to Makarova, K. S..

4 recordsLinked to original sources

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

Recruitment of CRISPR-Cas systems by Tn7-like transposons

A survey of bacterial and archaeal genomes shows that many Tn7-like transposons contain minimal type I-F CRISPR-Cas systems that consist of fused cas8f and cas5f, cas7f and cas6f genes, and a short CRISPR array. Additionally, several small groups of Tn7-like transposons encompass similarly truncated type I-B CRISPR-Cas systems. This gene composition of the transposon-associated CRISPR-Cas systems implies that they are competent for pre-crRNA processing yielding mature crRNAs and target binding but not target cleavage that is required for interference. Here we present phylogenetic analysis demonstrating that evolution of the CRISPR-Cas containing transposons included a single, ancestral capture of a type I-F locus and two independent instances of type I-B loci capture. We further show that the transposon-associated CRISPR arrays contain spacers homologous to plasmid and temperate phage sequences, and in some cases, chromosomal sequences adjacent to the transposon. A hypothesis is proposed that the transposon-encoded CRISPR-Cas systems generate displacement (R-loops) in the cognate DNA sites, targeting the transposon to these sites and thus facilitating their spread via plasmids and phages. This scenario fits the \"guns for hire\" concept whereby mobile genetic elements can capture host defense systems and repurpose them for different stages in the life cycle of the element.\n\nImportanceCRISPR-Cas is an adaptive immunity system that protects bacteria and archaea from mobile genetic elements. We present comparative genomic and phylogenetic analysis of degenerate CRISPR-Cas variants associated with distinct families of transposable elements and develop the hypothesis that such repurposed defense systems contribute to the transposable element propagation by facilitating transposition into specific sites. Such recruitment of defense systems by mobile elements supports the \"guns for hire\" concept under which the same enzymatic machineries can be alternately employed for transposon proliferation or host defense.

evolutionary biology↗

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↗

bioRxiv2016

Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentially regulated by accessory proteins Csx27 and Csx28

CRISPR-Cas adaptive immune systems defend microbes against foreign nucleic acids via RNA-guided endonucleases. Using a computational sequence database mining approach, we identify two Class 2 CRISPR-Cas systems (subtype VI-B) that lack Cas1 and Cas2 and encompass a single large effector protein, Cas13b, along with one of two previously uncharacterized associated proteins, Csx27 or Csx28. We establish that these CRISPR-Cas systems can achieve RNA interference when heterologously expressed. Through a combination of biochemical and genetic experiments, we show that Cas13b processes its own CRISPR array with short and long direct repeats, cleaves target RNA, and exhibits collateral RNase activity. Using an E. coli essential gene screen, we demonstrate that Cas13b has a double-sided protospacer-flanking sequence and elucidate RNA secondary structure requirements for targeting. We also find that Csx27 represses, whereas Csx28 enhances, Cas13b-mediated RNA interference. Characterization of these CRISPR systems creates opportunities to develop tools to manipulate and monitor cellular transcripts.

microbiology↗