Spacer acquisition by Type III CRISPR–Cas system during bacteriophage infection of Thermus thermophilus

Abstract

Type III CRISPR–Cas systems provide immunity to foreign DNA by targeting its transcripts. Target recognition activates RNases and DNases that may either destroy foreign DNA directly or elicit collateral damage inducing death of infected cells. While some Type III systems encode a reverse transcriptase to acquire spacers from foreign transcripts, most contain conventional spacer acquisition machinery found in DNA-targeting systems. We studied Type III spacer acquisition in phage-infected Thermus thermophilus, a bacterium that lacks either a standalone reverse transcriptase or its fusion to spacer integrase Cas1. Cells with spacers targeting a subset of phage transcripts survived the infection, indicating that Type III immunity does not operate through altruistic suicide. In the absence of selection spacers were acquired from both strands of phage DNA, indicating that no mechanism ensuring acquisition of RNA-targeting spacers exists. Spacers that protect the host from the phage demonstrate a very strong strand bias due to positive selection during infection. Phages that escaped Type III interference accumulated deletions of integral number of codons in an essential gene and much longer deletions in a non-essential gene. This and the fact that Type III immunity can be provided by plasmid-borne mini-arrays open ways for genomic manipulation of Thermus phages.

Comprehensive PAM prediction for CRISPR-Cas systems reveals evidence for spacer sharing, preferred strand targeting and conserved links with CRISPR repeats

“…This result demonstrates that there is selection in Type III systems for spacers to target the transcribed RNA. This selection can originate at the adaptation stage by dedicated adaptation machinery selecting from RNA/coding strands such as RT-Cas1 (Silas et al, 2016) or at the interference stage, where only functional RNA-targeting spacers are retained in the population (Artamonova et al, 2020). The strand biases we found are consistent with our curated CRISPR array orientation predictions, because an incorrect CRISPR array orientation prediction would obscure strand specific targeting.…”

SCOPE: Flexible targeting and stringent CARF activation enables type III CRISPR-Cas diagnostics

“…Recent advancements in our understanding of type III CRISPR-Cas systems have highlighted that they have unique mechanistic features compared to other CRISPR-Cas systems. Examples of this include the requirement for reverse-transcriptase activity for some type III systems during the adaptation phase [45]–[47] and the potentially large signaling network mediated by cOA molecules in the interference phase [27]–[29][32][45]–[47]. Furthermore, type III systems are exceptional in the sense that they are the only CRISPR-Cas system characterized to date capable of targeting both RNA (guide dependent) and DNA (collateral).…”

PAM-repeat associations and spacer selection preferences in single and co-occurring CRISPR-Cas systems

“…However, in other situations, the observed patterns might have been the result of selection for a working TIM. For example, most of the spacers selected for in RNA targeting systems were found to be acquired at random [20], even though spacers present in natural CRISPR arrays often show a bias towards the coding strand [21,22], suggesting that the bias emerged from effective interference spacers through natural selection. On the other hand, there are systems that contain a reverse transcriptase fused to Cas1 (RT-Cas1) [23] which can already select spacers from the correct strand.…”