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Title: RNA-dependent RNA targeting by CRISPR-Cas9

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. In conclusion, 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:
ORCiD logo [1] ;  [2] ;  [3] ;  [4] ; ORCiD logo [5]
  1. Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology
  2. Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology; Univ. of Michigan, Ann Arbor, MI (United States). Dept. of Medicinal Chemistry
  3. Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology; Global Blood Therapeutics, San Francisco, CA (United States)
  4. Sandia National Lab. (SNL-CA), Livermore, CA (United States). Biotechnology and Bioengineering Dept.
  5. Univ. of California, Berkeley, CA (United States). Innovative Genomics Inst. and Dept. of Molecular and Cell Biology and Dept. of Chemistry; Howard Hughes Medical Inst., Chevy Chase, MD (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division
Publication Date:
Grant/Contract Number:
AC02-05CH11231; MCB-1244557; NA0003525; S10RR029668; S10RR027303
Type:
Published Article
Journal Name:
eLife
Additional Journal Information:
Journal Volume: 7; Journal Issue: none; Related Information: © Strutt et al.; Journal ID: ISSN 2050-084X
Publisher:
eLife Sciences Publications, Ltd.
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE National Nuclear Security Administration (NNSA); National Science Foundation (NSF); Howard Hughes Medical Inst., Chevy Chase, MD (United States); The Paul G. Allen Frontiers Group, Univ. of California, Berkeley, CA (United States); National Institutes of Health (NIH)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
OSTI Identifier:
1415873
Alternate Identifier(s):
OSTI ID: 1415874; OSTI ID: 1433112

Strutt, Steven C., Torrez, Rachel M., Kaya, Emine, Negrete, Oscar A., and Doudna, Jennifer A.. RNA-dependent RNA targeting by CRISPR-Cas9. United States: N. p., Web. doi:10.7554/eLife.32724.
Strutt, Steven C., Torrez, Rachel M., Kaya, Emine, Negrete, Oscar A., & Doudna, Jennifer A.. RNA-dependent RNA targeting by CRISPR-Cas9. United States. doi:10.7554/eLife.32724.
Strutt, Steven C., Torrez, Rachel M., Kaya, Emine, Negrete, Oscar A., and Doudna, Jennifer A.. 2018. "RNA-dependent RNA targeting by CRISPR-Cas9". United States. doi:10.7554/eLife.32724.
@article{osti_1415873,
title = {RNA-dependent RNA targeting by CRISPR-Cas9},
author = {Strutt, Steven C. and Torrez, Rachel M. and Kaya, Emine and Negrete, Oscar A. and Doudna, Jennifer A.},
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. In conclusion, 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 = none,
volume = 7,
place = {United States},
year = {2018},
month = {1}
}

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Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression
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Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems
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DNA targeting specificity of RNA-guided Cas9 nucleases
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A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity
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Multiplex Genome Engineering Using CRISPR/Cas Systems
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RNA-guided editing of bacterial genomes using CRISPR-Cas systems
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