On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires

Abstract

Cas1 integrase is the key enzyme of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas adaptation module that mediates acquisition of spacers derived from foreign DNA by CRISPR arrays. In diverse bacteria, the cas1 gene is fused (or adjacent) to a gene encoding a reverse transcriptase (RT) related to group II intron RTs. An RT-Cas1 fusion protein has been recently shown to enable acquisition of CRISPR spacers from RNA. Phylogenetic analysis of the CRISPR-associated RTs demonstrates monophyly of the RT-Cas1 fusion, and coevolution of the RT and Cas1 domains. Nearly all such RTs are present within type III CRISPR-Cas loci, but their phylogeny does not parallel the CRISPR-Cas type classification, indicating that RT-Cas1 is an autonomous functional module that is disseminated by horizontal gene transfer and can function with diverse type III systems. To compare the sequence pools sampled by RT-Cas1-associated and RT-lacking CRISPR-Cas systems, we obtained samples of a commercially grown cyanobacterium—Arthrospira platensis. Sequencing of the CRISPR arrays uncovered a highly diverse population of spacers. Spacer diversity was particularly striking for the RT-Cas1-containing type III-B system, where no saturation was evident even with millions of sequences analyzed. In contrast, analysis of the RT-lacking type III-D system yielded a highly diverse pool but reached a point where fewer novel spacers were recovered as sequencing depth was increased. Matches could be identified for a small fraction of the non-RT-Cas1-associated spacers, and for only a single RT-Cas1-associated spacer. Thus, the principal source(s) of the spacers, particularly the hypervariable spacer repertoire of the RT-associated arrays, remains unknown.

Targeted transposition with Tn7 elements: safe sites, mobile plasmids, CRISPR/Cas and beyond

“…It has even been suggested that the first CRISPR/Cas system could have been used for targeting transposition with ancient casposon elements, a process that later re‐evolved with Tn7‐like elements (Koonin and Makarova, ). In another example of exchange, spacer acquisition in one subtype within the type III CRISPR/Cas systems gained the capacity to harvest spacer information from RNA through the apparent capture of reverse transcriptase activity from Group II introns (Silas et al , ; Silas et al , ). Finally, the Cas12 effector proteins of type V CRISPR/Cas systems appears to have been derived from a RuvC‐like nuclease activity from IS605‐like transposons on multiple occasions (Koonin and Makarova, ).…”

An atypical CRISPR-Cas locus in Symbiobacterium thermophilum flanked by a transposase, a reverse transcriptase, the endonuclease MutS2 and a putative Cas9-like protein

“…Moreover, these work seeded on known CRISPR-Cas loci, and thus would not have looked at the locus described here. The spacer sequences here (Fig 1a) could not identify any known organism, echoing the observation that 'the vast majority of RT-linked CRISPR spacers come from a distinct sequence pool, the nature of which remains enigmatic' [29].…”

“…In many Type III CRISPR systems, a reverse transcriptase (RT) is fused to a Cas1, which in the marine bacterium Marinomonas mediterranea, was shown to mediate the in vivo acquisition of RNA spacers [28]. Two recent studies looked at the evolutionary origins of RT-CRISPR, and looked mostly at RT-Cas1 fusions [29,30]. Moreover, these work seeded on known CRISPR-Cas loci, and thus would not have looked at the locus described here.…”

“…Intriguingly, the 1357 is uncannily close to length of known Cas9 proteins (1368 for Streptococcus pyogenes) -Symbiobacterium thermophilum does not have a Cas9 CRISPR-Cas locus. Finally, a group II intron reverse transcriptase (RT) downstream of the repeat suggest that this locus is from a family of enigmatic RT-CRISPR systems [29], while the locus also has transposases that explain the CRISPR-Cas genesis.…”

“…The presence of a reverse transcriptase (RT, WP 011194877.1) downstream of the repeat indicates this locus is a RT-CRISPR that integrates RNA (after the RT transcribes it into DNA) [28][29][30], possibly using a proximal transposase [31,32].…”