@article{70263c3bcb544ed3a7ed7fc1af73f3bf,
title = "Programmable Inhibition and Detection of RNA Viruses Using Cas13",
abstract = "The CRISPR effector Cas13 could be an effective antiviral for single-stranded RNA (ssRNA) viruses because it programmably cleaves RNAs complementary to its CRISPR RNA (crRNA). Here, we computationally identify thousands of potential Cas13 crRNA target sites in hundreds of ssRNA viral species that can potentially infect humans. We experimentally demonstrate Cas13's potent activity against three distinct ssRNA viruses: lymphocytic choriomeningitis virus (LCMV); influenza A virus (IAV); and vesicular stomatitis virus (VSV). Combining this antiviral activity with Cas13-based diagnostics, we develop Cas13-assisted restriction of viral expression and readout (CARVER), an end-to-end platform that uses Cas13 to detect and destroy viral RNA. We further screen hundreds of crRNAs along the LCMV genome to evaluate how conservation and target RNA nucleotide content influence Cas13's antiviral activity. Our results demonstrate that Cas13 can be harnessed to target a wide range of ssRNA viruses and CARVER's potential broad utility for rapid diagnostic and antiviral drug development. Freije et al. demonstrate that Cas13 can be programmed to target and destroy the genomes of diverse mammalian single-stranded RNA viruses. They identify design principles for efficient Cas13 targeting of viral RNA and create companion Cas13-based diagnostics to rapidly measure the effects of Cas13 targeting.",
keywords = "Arenavirus, CRISPR, Cas13, Cas13-based detection, RNA interference, RNA viruses, antiviral, crRNA design, influenza virus, multiplexing",
author = "Freije, {Catherine A.} and Cameron Myhrvold and Boehm, {Chloe K.} and Lin, {Aaron E.} and Welch, {Nicole L.} and Amber Carter and Metsky, {Hayden C.} and Luo, {Cynthia Y.} and Abudayyeh, {Omar O.} and Gootenberg, {Jonathan S.} and Yozwiak, {Nathan L.} and Feng Zhang and Sabeti, {Pardis C.}",
note = "Funding Information: We thank J. de la Torre for providing the BHK-21 cell line and rLCMV-GFP viral stocks, protocols, and thoughtful discussions and reading of the manuscript. We thank D. Lingwood and M. Sangesland for providing the MDCK cell line and IAV viral stocks and protocols. We thank S. Whelan and L. Gehrke for providing rVSV-GFP viral stocks, BsrT7 cell line, and protocols. We thank M. Tabebordbar, A. Piantadosi, K. Barnes, K. Siddle, S. Wohl, E. Brown, and other Sabeti lab members for discussions and thoughtful manuscript feedback. Funding was provided from HHMI , NIH grant U19AI110818 , and Defense Advanced Research Projects Agency (DARPA) grant D18AC00006 . The views, opinions, and/or findings expressed should not be interpreted as representing the official views or policies of the Department of Defense or the US government. This study has been approved for public release; distribution is unlimited. F.Z. is supported by NIH grants 1R01-HG009761 , 1R01-MH110049 , and 1DP1-HL141201 ; HHMI ; the New York Stem Cell , Allen , and Vallee foundations; the Tan-Yang Center at MIT ; and J. and P. Poitras and R. Metcalfe . F.Z. is a New York Stem Cell Foundation-Robertson Investigator. All data are available in the article or the supplemental materials. Funding Information: We thank J. de la Torre for providing the BHK-21 cell line and rLCMV-GFP viral stocks, protocols, and thoughtful discussions and reading of the manuscript. We thank D. Lingwood and M. Sangesland for providing the MDCK cell line and IAV viral stocks and protocols. We thank S. Whelan and L. Gehrke for providing rVSV-GFP viral stocks, BsrT7 cell line, and protocols. We thank M. Tabebordbar, A. Piantadosi, K. Barnes, K. Siddle, S. Wohl, E. Brown, and other Sabeti lab members for discussions and thoughtful manuscript feedback. Funding was provided from HHMI, NIH grant U19AI110818, and Defense Advanced Research Projects Agency (DARPA) grant D18AC00006. The views, opinions, and/or findings expressed should not be interpreted as representing the official views or policies of the Department of Defense or the US government. This study has been approved for public release; distribution is unlimited. F.Z. is supported by NIH grants 1R01-HG009761, 1R01-MH110049, and 1DP1-HL141201; HHMI; the New York Stem Cell, Allen, and Vallee foundations; the Tan-Yang Center at MIT; and J. and P. Poitras and R. Metcalfe. F.Z. is a New York Stem Cell Foundation-Robertson Investigator. All data are available in the article or the supplemental materials. C.A.F. and C.M. conceived the study and wrote the paper. P.C.S. supervised the study (with assistance from N.L.Y.). C.A.F. C.M. C.K.B. A.E.L. N.L.W. A.C. and C.Y.L. performed experiments and data analysis. H.C.M. developed the crRNA target site search software. O.O.A. and J.S.G. designed transfection protocols and provided reagents and technical assistance (supervised by F.Z.). All authors reviewed the manuscript. C.A.F. C.M. P.C.S. O.O.A. J.S.G. and F.Z. are co-inventors on patent applications filed by the Broad Institute relating to work in this study. O.O.A. J.S.G. F.Z. and P.C.S. are co-founders of Sherlock Biosciences. F.Z. is a cofounder and advisor of Beam Therapeutics. O.O.A. and J.S.G. are advisors for Beam Therapeutics. Publisher Copyright: {\textcopyright} 2019 The Authors",
year = "2019",
month = dec,
day = "5",
doi = "10.1016/j.molcel.2019.09.013",
language = "English (US)",
volume = "76",
pages = "826--837.e11",
journal = "Molecular Cell",
issn = "1097-2765",
publisher = "Cell Press",
number = "5",
}