A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression

Abstract

CRISPR-Cas systems provide their prokaryotic hosts with acquired immunity against viruses and other foreign genetic elements, but how these systems are regulated to prevent auto-immunity is poorly understood. In type II CRISPR-Cas systems, a transactivating CRISPR RNA (tracrRNA) scaffold functions together with a CRISPR RNA (crRNA) guide to program Cas9 for the recognition and cleavage of foreign DNA targets. Here, we show that a long-form tracrRNA performs an unexpected second function by folding into a natural single guide that directs Cas9 to transcriptionally repress its own promoter. Further, we demonstrate that Pcas9 serves as a critical regulatory node; de-repression causes a dramatic induction of Cas genes, crRNAs and tracrRNAs resulting in a 3,000-fold increase in immunization rates against unrecognized viruses. Heightened immunity comes at the cost of increased auto-immune toxicity, demonstrating the critical importance of the controller. Using a bioinformatic analysis, we provide evidence that tracrRNA-mediated autoregulation is widespread in type II CRISPR-Cas systems. Collectively, we unveil a new paradigm for the intrinsic regulation of CRISPR-Cas systems by natural single guides, which may facilitate the frequent horizontal transfer of these systems into new hosts that have not yet evolved their own regulatory strategies.

Distinct subcellular localization of a Type I CRISPR complex and the Cas3 nuclease in bacteria

“…In contrast to the Cas3 nuclease, localization of Cas9 in the nucleoid could pose autoimmunity risks. In fact, a recent study showed that uncontrolled induction Cas9 in S. pyogenes, which occurs in the absence of a natural single guide RNA, results in increased self-targeting and auto immunity (Workman et al, 2020). Anecdotally, we have also observed that activity of Listeria monocytogenes Cas9 is very low, at least in laboratory conditions (Rauch et al, 2017).…”

“…The molecular basis for most of these alternative functions of Cas proteins are not well understood. A recent study showing SpyCas9 can repress its own promoter using a natural single guide opens the possibility that CRISPR-Cas systems can also function as intrinsic transcriptional regulators (Workman et al, 2020). It is therefore possible that crRNAs generated from degenerate selftargeting spacers might direct CRISPR-Cas enzymes in regulating host genes.…”

Epidemiological and evolutionary consequences of CRISPR-Cas reactivity

“…However, this is not what is observed in nature, when CRISPR-Cas reactivity is usually low (Hynes et al, 2017). In addition, scientists can easily generate CRISPR-Cas systems with higher reactivities (Heler et al, 2017; Levy et al, 2015; Workman et al, 2020), so their absence is not the result of evolutionary constraints.…”

“…To the best of our knowledge, the precise relationship between CRISPR-Cas reactivity and CRISPR-Cas autoimmunity is unknown. However, we know that an increase in reactivity results in higher autoimmunity (Levy et al, 2015; Workman et al, 2020). In addition, there is limited evidence that an increase in reactivity is proportional to autoimmunity (Levy et al, 2015).…”

“…to the acquisition of a spacer that targets a chromosomal sequence, which triggers its degradation and this is thought to be often lethal (Stern et al, 2010; Jiang et al, 2013; Vercoe et al, 2013; Gomaa et al, 2014; Wimmer and Beisel, 2020). Importantly, the level of autoimmunity of a CRISPR-Cas system is related to its reactivity (Levy et al, 2015; Workman et al, 2020). Therefore, it is likely that CRISPR-Cas systems face a similar control challenge as vertebrate adaptive immune systems: too much reactivity increases autoimmunity but low levels of reactivity decrease the efficiency of the immune response (Weissman et al, 2020).…”