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Title: RNA-dependent RNA targeting by CRISPR-Cas9.

Abstract

Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo. We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. These results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications.

Authors:
 [1];  [1];  [1];  [2];  [1]
  1. Univ. of California, Berkeley, CA (United States)
  2. Sandia National Lab. (SNL-CA), Livermore, CA (United States)
Publication Date:
Research Org.:
Sandia National Lab. (SNL-CA), Livermore, CA (United States)
Sponsoring Org.:
USDOE National Nuclear Security Administration (NNSA)
OSTI Identifier:
1477327
Report Number(s):
SAND-2018-10613J
Journal ID: ISSN 2050-084X; 668201
Grant/Contract Number:  
AC04-94AL85000
Resource Type:
Accepted Manuscript
Journal Name:
eLife
Additional Journal Information:
Journal Volume: 7; Journal ID: ISSN 2050-084X
Publisher:
eLife Sciences Publications, Ltd.
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Strutt, Steven, Torrez, Rachel, Kaya, Emine, Negrete, Oscar, and Doudna, Jennifer. RNA-dependent RNA targeting by CRISPR-Cas9.. United States: N. p., 2018. Web. doi:/10.7554/eLife.32724.
Strutt, Steven, Torrez, Rachel, Kaya, Emine, Negrete, Oscar, & Doudna, Jennifer. RNA-dependent RNA targeting by CRISPR-Cas9.. United States. doi:/10.7554/eLife.32724.
Strutt, Steven, Torrez, Rachel, Kaya, Emine, Negrete, Oscar, and Doudna, Jennifer. Fri . "RNA-dependent RNA targeting by CRISPR-Cas9.". United States. doi:/10.7554/eLife.32724. https://www.osti.gov/servlets/purl/1477327.
@article{osti_1477327,
title = {RNA-dependent RNA targeting by CRISPR-Cas9.},
author = {Strutt, Steven and Torrez, Rachel and Kaya, Emine and Negrete, Oscar and Doudna, Jennifer},
abstractNote = {Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo. We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. These results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications.},
doi = {/10.7554/eLife.32724},
journal = {eLife},
number = ,
volume = 7,
place = {United States},
year = {2018},
month = {1}
}

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