Programmable Inhibition and Detection of RNA Viruses Using Cas13

Catherine A Freije; Cameron Myhrvold; Chloe K Boehm; Aaron E Lin; Nicole L Welch; Amber Carter; Hayden C Metsky; Cynthia Y Luo; Omar O Abudayyeh; Jonathan S Gootenberg; Nathan L Yozwiak; Feng Zhang; Pardis C Sabeti

doi:10.1016/j.molcel.2019.09.013

Programmable Inhibition and Detection of RNA Viruses Using Cas13

Mol Cell. 2019 Dec 5;76(5):826-837.e11. doi: 10.1016/j.molcel.2019.09.013. Epub 2019 Oct 10.

Authors

Affiliations

¹ Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA 02142, USA; PhD Program in Virology, Division of Medical Sciences, Harvard Medical School, Boston, MA 02115, USA. Electronic address: cfreije@broadinstitute.org.
² Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA 02142, USA; Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA. Electronic address: cmyhrvol@broadinstitute.org.
³ Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA 02142, USA.
⁴ Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA 02142, USA; PhD Program in Virology, Division of Medical Sciences, Harvard Medical School, Boston, MA 02115, USA.
⁵ Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA 02142, USA; Department of Electrical Engineering and Computer Science, MIT, Cambridge, MA 02142, USA.
⁶ Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA 02142, USA; Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA.
⁷ Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, MIT, Cambridge, MA 02139, USA; Department of Biological Engineering, MIT, Cambridge, MA 02139, USA; Department of Health Sciences and Technology, MIT, Cambridge, MA 02139, USA.
⁸ Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, MIT, Cambridge, MA 02139, USA; Department of Biological Engineering, MIT, Cambridge, MA 02139, USA; Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
⁹ Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, MIT, Cambridge, MA 02139, USA; Department of Biological Engineering, MIT, Cambridge, MA 02139, USA; Howard Hughes Medical Institute (HHMI), Chevy Chase, MD 20815, USA.
¹⁰ Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA 02142, USA; PhD Program in Virology, Division of Medical Sciences, Harvard Medical School, Boston, MA 02115, USA; Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA; Howard Hughes Medical Institute (HHMI), Chevy Chase, MD 20815, USA; Department of Immunology and Infectious Disease, T.H. Chan Harvard School of Public Health, Boston, MA 02115, USA. Electronic address: pardis@broadinstitute.org.

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.

Keywords: Arenavirus; CRISPR; Cas13; Cas13-based detection; RNA interference; RNA viruses; antiviral; crRNA design; influenza virus; multiplexing.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

A549 Cells
Animals
CRISPR-Associated Proteins / genetics
CRISPR-Associated Proteins / metabolism*
CRISPR-Cas Systems*
Chlorocebus aethiops
Clustered Regularly Interspaced Short Palindromic Repeats*
Dogs
Escherichia coli / enzymology
Escherichia coli / genetics
Gene Targeting / methods*
HEK293 Cells
Humans
Madin Darby Canine Kidney Cells
RNA Stability*
RNA Viruses / enzymology*
RNA Viruses / genetics
RNA, Viral / genetics
RNA, Viral / metabolism*
Vero Cells

Substances

CRISPR-Associated Proteins
RNA, Viral

Abstract

Publication types

MeSH terms

Substances

Grants and funding