PT  - JOURNAL ARTICLE
AU  - Rachael E. Workman
AU  - Teja Pammi
AU  - Binh T. K. Nguyen
AU  - Leonardo W. Graeff
AU  - Erika Smith
AU  - Suzanne M. Sebald
AU  - Marie J. Stoltzfus
AU  - Joshua W. Modell
TI  - A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression
AID  - 10.1101/2020.05.21.102756
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.05.21.102756
4099  - http://biorxiv.org/content/early/2020/05/21/2020.05.21.102756.short
4100  - http://biorxiv.org/content/early/2020/05/21/2020.05.21.102756.full
AB  - 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.Competing Interest StatementThe authors have declared no competing interest.