The CRISPR Spacer Space Is Dominated by Sequences from Species-Specific Mobilomes

Abstract

Clustered regularly interspaced short palindromic repeats and CRISPRassociated protein (CRISPR-Cas) systems store the memory of past encounters with foreign DNA in unique spacers that are inserted between direct repeats in CRISPR arrays. For only a small fraction of the spacers, homologous sequences, called protospacers, are detectable in viral, plasmid, and microbial genomes. 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 we 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%, originated from viral genomes, including proviruses, and among the rest, the most common source was genes that are integrated into microbial chromosomes but are 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 showed a nearly perfect correlation and were almost identical. Given the near absence of self-targeting spacers, these findings are most compatible with the possibility that the spacers, including the dark matter, are derived almost completely from the species-specific microbial mobilomes. IMPORTANCEThe principal function of CRISPR-Cas systems is thought to be protection of bacteria and archaea against viruses and other parasitic genetic elements. The CRISPR defense function is mediated by sequences from parasitic elements, known as spacers, that are inserted into CRISPR arrays and then transcribed and employed as guides to identify and inactivate the cognate parasitic genomes. However, only a small fraction of the CRISPR spacers match any sequences in the current databases, and of these, only a minority correspond to known parasitic elements. We show that nearly all spacers with matches originate from viral or plasmid genomes that are either free or have been integrated into the host genome. We further demonstrate that spacers with no matches have the same properties as those of identifiable origins, strongly suggesting that all spacers originate from mobile elements.KEYWORDS CRISPR-Cas, bacteriophages, mobilome, oligonucleotide composition, spacer acquisition C RISPR-Cas (clustered regularly interspaced palindromic repeats and CRISPRassociated proteins) systems are adaptive (acquired) immune systems of archaea and bacteria that store memory of past encounters with foreign DNA in unique spacer Citation Shmakov SA, Sitnik V, Makarova KS, Wolf YI, Severinov KV, Koonin EV. 2017. The CRISPR spacer space is dominated by sequences from species-specific mobilomes.

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

“…6 Supplementary Data 3 and 4). In agreement with previous reports 22 , all CRISPR-Cas types revealed a targeting preference for conserved, and frequently plasmid-borne, genes; e.g. conjugative transfer machinery genes ( Supplementary Fig.…”

“…Consistent with earlier results 1,8 , only a small fraction of spacers yielded significant matches: ~12% and ~7%, for type IV and non-type IV, respectively ( Fig. 3a, Supplementary Table 4 and Supplementary Data 3 and 4), which reflects the current undersampling of the microbiome 22 .…”

“…In agreement with previous work, we found that the newly identified type IV loci are primarily encoded by prokaryotic MGEs, most of which are predicted to be plasmids ( Figure 2). Notably, given the current limited sequence information covering the "dark matter" of the mobilome 22,35,36 , our findings likely underestimate the true diversity and distribution of these systems. Future comparative genomic characterizations will clearly benefit from including metagenomic sequence datasets and the continuing global effort to sample the meta-mobilome.…”

“…On the other hand, nontype IV subtypes overall exhibited the previously reported strong preference for viral targets ( Fig. 3a., Supplementary Table 4) 1,22 . Given that the type IV and non-type IV spacer contents investigated here originate from the same cellular environments, the results strongly underline an anti-plasmid function for type IV systems.…”

Cylindrospermopsis raciborskii Virus and host: genomic characterization and ecological relevance

“…The N‐1 array contains four spacers with 37 bp DRs. Like CrV, the N‐1 spacers had no significant matches in the nr nucleotide database, a common observation across the entirety of CRISPR spacers analysed (Shmakov et al ., ). No cas genes were found in the N‐1 genome.…”