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. 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:
-
[1];
[5]
- Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology
- 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
- Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology; Global Blood Therapeutics, San Francisco, CA (United States)
- Sandia National Lab. (SNL-CA), Livermore, CA (United States). Biotechnology and Bioengineering Dept.
- 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:
- Research Org.:
- Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); Sandia National Lab. (SNL-CA), Livermore, 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)
- OSTI Identifier:
- 1415873
- Alternate Identifier(s):
- OSTI ID: 1415874; OSTI ID: 1433112; OSTI ID: 1477327
- Report Number(s):
- SAND-2018-10613J
Journal ID: ISSN 2050-084X; e32724
- Grant/Contract Number:
- AC02-05CH11231; MCB-1244557; NA0003525; S10RR029668; S10RR027303; AC04-94AL85000
- Resource Type:
- Published Article
- Journal Name:
- eLife
- Additional Journal Information:
- Journal Name: eLife 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; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
Citation Formats
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., 2018.
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. https://doi.org/10.7554/eLife.32724
Strutt, Steven C., Torrez, Rachel M., Kaya, Emine, Negrete, Oscar A., and Doudna, Jennifer A. Fri .
"RNA-dependent RNA targeting by CRISPR-Cas9". United States. https://doi.org/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 = ,
volume = 7,
place = {United States},
year = {Fri Jan 05 00:00:00 EST 2018},
month = {Fri Jan 05 00:00:00 EST 2018}
}
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https://doi.org/10.7554/eLife.32724
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