Cas13-induced cellular dormancy prevents the rise of CRISPR-resistant bacteriophage

Abstract

Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci of prokaryotes are composed of 30-40 bp repeats separated by equally short sequences of plasmid and bacteriophage origin known as spacers 1 – 3 . Spacers are transcribed and processed into short CRISPR RNAs (crRNAs) that are used as guides by CRISPR-associated (Cas) nucleases to recognize and destroy complementary sequences (known as protospacers) within invaders 4 , 5 . In contrast to most Cas nucleases which destroy the invader’s DNA 4 – 7 , the type VI effector nuclease Cas13 employs RNA guides to locate complementary transcripts and catalyze both sequence-specific cis -, and non-specific trans -RNA cleavage 8 . While it has been hypothesized that Cas13 naturally defends against RNA phages 8 , type VI spacer sequences have exclusively been found to match the genomes of double-stranded DNA (dsDNA) phages 9 , 10 , suggesting that Cas13 can provide immunity against these invaders. However, whether and how Cas13 utilizes the cis - and/or trans -RNA cleavage activities in defending against dsDNA phages is not understood. Here we show that trans -cleavage of transcripts halts the growth of the host cell and results in the abortion of the infectious cycle. This depletes the phage population and provides herd immunity to uninfected bacteria. Phages harboring target mutations, which easily evade DNA-targeting CRISPR systems 11 – 13 , are also depleted due to the activation of Cas13 by co-infecting wild type phages. Thus, by acting on the host rather than directly targeting the virus, type VI CRISPR systems not only provide robust defense against DNA phages but also prevent outbreaks of CRISPR-resistant phage.

Programmable RNA Targeting using CasRx in Flies

“…Through this study, we identified two main factors contributing to CasRx-mediated lethality: (i) the catalytic activity of the CasRx HEPN domains, as lethality and tissue necrosis phenotypes were eliminated in dCasRx compared to CasRx crosses, and (ii) the presence of the target transcript resulting in on-target cleavage, as lethality was only observed when crossing Ubiq-CasRxexpressing flies to gRNA GFP -expressing flies. These results recapitulate previous mechanistic analyses of CasRx and other Cas13 ribonucleases, demonstrating that collateral off-target activity following targeted transcript cleavage is a native feature of Cas13 ribonucleases [2,4,[7][8][9]45]. While this feature may not be desirable for generating tools for targeting specific transcripts of genes, this may be useful for generating sensors that get activated in response to a target transcript (e.g.…”

“…Specifically, RNA targeting was demonstrated with ubiquitous, inducible and tissue specific CasRx expression systems against native and synthetic RNA targets, which are prerequisites for enabling comprehensive studies of gene function. However, we did also consistently observe both cellular toxicity from the ubiquitous expression of CasRx and dCasRx as we could not generate homozygous strains for either, and unexpected lethality and tissue necrosis, presumably due collateral off target effects which have been a feature previously observed for many CRISPR ribonucleases including CasRx [2,4,[7][8][9]45]. Nevertheless, in both bidirectional and Gal4/UAS crosses, we were able to obtain visible phenotypes as well as quantitative evidence (e.g., RNAseq data demonstrating a reduction in target gene expression) indicating that the CasRx is capable of targeting and degrading target RNA in flies.…”

Pervasive off-target and double-stranded DNA nicking by CRISPR-Cas12a

“…In CRISPR-Cas systems like type III and type VI, Cas nucleases can be activated for nonspecific cleavage of RNA (Hille et al 2018). Perhaps, this is a means to prevent phage proliferation by initiating programmed cell death (PCD), abortive infection or dormancy in order to save the bacterial population (Koonin and Zhang 2017;Meeske, Nakandakari-Higa, and Marraffini 2019). Further studies are required to investigate the cost-benefit relation of such nonspecific activities of Cas12a to the bacteria.…”

Structure and mechanism of a Type III CRISPR defence DNA nuclease activated by cyclic oligoadenylate

“…In addition to the cOA activated, non-specific ribonucleases described above for type III systems, type VI (Cas13) effectors provide effective immunity against mobile genetic elements by degrading RNA non-specifically 32,33 . The mechanism for this immunity was recently shown to result in degradation of host, rather than virus, RNA, driving infected cells into dormancy and thus preventing the spread of infection 34 . DNA is also a target for collateral degradation.…”