@article{e3b3d7b762ce402ab96a4e2f96045bc2,
title = "A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression",
abstract = "CRISPR-Cas systems provide prokaryotes with acquired immunity against viruses and plasmids, but how these systems are regulated to prevent autoimmunity is poorly understood. Here, we show that in the S. pyogenes CRISPR-Cas system, a long-form transactivating CRISPR RNA (tracr-L) folds into a natural single guide that directs Cas9 to transcriptionally repress its own promoter (Pcas). Further, we demonstrate that Pcas serves as a critical regulatory node. De-repression causes a dramatic 3,000-fold increase in immunization rates against viruses; however, heightened immunity comes at the cost of increased autoimmune toxicity. Using bioinformatic analyses, we provide evidence that tracrRNA-mediated autoregulation is widespread in type II-A 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.",
keywords = "CRISPR-Cas, Cas9, autoimmunity, bacterial regulation, bacteriophage, sgRNA, tracr-L, tracrRNA",
author = "Workman, {Rachael E.} and Teja Pammi and Nguyen, {Binh T.K.} and Graeff, {Leonardo W.} and Erika Smith and Sebald, {Suzanne M.} and Stoltzfus, {Marie J.} and Euler, {Chad W.} and Modell, {Joshua W.}",
note = "Funding Information: We would like to thank Michael Laub, Luciano Marraffini, Geraldine Seydoux, and Samuel Sternberg for their comments on the manuscript. We thank Nicholas Keith for lending bioinformatic expertise. We thank Karole D'Orazio and members of Rachel Green's and Scott Bailey's labs for sharing technical assistance and reagents for in vitro experiments. We thank Jeremy Nathans{\textquoteright} and Carol Greider's lab for sharing reagents and equipment. We thank David Mohr and the GRCF High Throughput Sequencing Center for assistance with NGS experiments. Funding was provided by a startup package from the Johns Hopkins School of Medicine. R.E.W. T.P. B.T.K.N. L.W.G. and J.W.M. designed and executed the research studies. E.S. S.M.S. and M.J.S. assisted with plasmid construction. C.W.E. constructed the S. pyogenes Δtr-L strain and assisted in Streptococcal experimental design. R.E.W. and J.W.M. wrote the manuscript. The authors declare no competing interests. Publisher Copyright: {\textcopyright} 2020 Elsevier Inc.",
year = "2021",
month = feb,
day = "4",
doi = "10.1016/j.cell.2020.12.017",
language = "English (US)",
volume = "184",
pages = "675--688.e19",
journal = "Cell",
issn = "0092-8674",
publisher = "Cell Press",
number = "3",
}