RNA-dependent RNA targeting by CRISPR-Cas9

Strutt, Steven C.; Torrez, Rachel M.; Kaya, Emine; Negrete, Oscar A.; Doudna, Jennifer A.

doi:10.7554/eLife.32724

Title: RNA-dependent RNA targeting by CRISPR-Cas9

Journal Article · Fri Jan 05 00:00:00 EST 2018 · eLife

DOI:https://doi.org/10.7554/eLife.32724· OSTI ID:1415873

^[1]; Torrez, Rachel M. ^[2]; Kaya, Emine ^[3]; Negrete, Oscar A. ^[4];

^[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

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.

View Journal Article

Cite

Export

Save

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States)

Sponsoring Organization:: 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)

Grant/Contract Number:: AC02-05CH11231; MCB-1244557; NA0003525; S10RR029668; S10RR027303; AC04-94AL85000

OSTI ID:: 1415873

Alternate ID(s):: OSTI ID: 1415874; OSTI ID: 1433112; OSTI ID: 1477327

Report Number(s):: SAND-2018-10613J; e32724

Journal Information:: eLife, Journal Name: eLife Vol. 7; ISSN 2050-084X

Publisher:: eLife Sciences Publications, Ltd.Copyright Statement

Country of Publication:: United States

Language:: English

Citation Metrics:

Cited by: 119 works

Citation information provided by
Web of Science

References (65)

The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems Chylinski, Krzysztof; Le Rhun, Anaïs; Charpentier, Emmanuelle RNA Biology, Vol. 10, Issue 5 https://doi.org/10.4161/rna.24321	journal	February 2013
Benefits and Challenges with Applying Unique Molecular Identifiers in Next Generation Sequencing to Detect Low Frequency Mutations Kou, Ruqin; Lam, Ham; Duan, Hairong PLOS ONE, Vol. 11, Issue 1 https://doi.org/10.1371/journal.pone.0146638	journal	January 2016
Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes Ma, Enbo; Harrington, Lucas B.; O’Connell, Mitchell R. Molecular Cell, Vol. 60, Issue 3 https://doi.org/10.1016/j.molcel.2015.10.030	journal	November 2015
Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression Qi, Lei S.; Larson, Matthew H.; Gilbert, Luke A. Cell, Vol. 152, Issue 5, p. 1173-1183 https://doi.org/10.1016/j.cell.2013.02.022	journal	February 2013
Programmable RNA recognition and cleavage by CRISPR/Cas9 O’Connell, Mitchell R.; Oakes, Benjamin L.; Sternberg, Samuel H. Nature, Vol. 516, Issue 7530 https://doi.org/10.1038/nature13769	journal	September 2014
Cutadapt removes adapter sequences from high-throughput sequencing reads Martin, Marcel EMBnet.journal, Vol. 17, Issue 1 https://doi.org/10.14806/ej.17.1.200	journal	May 2011
Sushi.R: flexible, quantitative and integrative genomic visualizations for publication-quality multi-panel figures Phanstiel, D. H.; Boyle, A. P.; Araya, C. L. Bioinformatics, Vol. 30, Issue 19 https://doi.org/10.1093/bioinformatics/btu379	journal	June 2014
Hairpins under tension: RNA versus DNA Bercy, Mathilde; Bockelmann, Ulrich Nucleic Acids Research https://doi.org/10.1093/nar/gkv860	journal	August 2015
Biology and Applications of CRISPR Systems: Harnessing Nature’s Toolbox for Genome Engineering Wright, Addison V.; Nuñez, James K.; Doudna, Jennifer A. Cell, Vol. 164, Issue 1-2 https://doi.org/10.1016/j.cell.2015.12.035	journal	January 2016
New Algorithms and Methods to Estimate Maximum-Likelihood Phylogenies: Assessing the Performance of PhyML 3.0 Guindon, Stéphane; Dufayard, Jean-François; Lefort, Vincent Systematic Biology, Vol. 59, Issue 3 https://doi.org/10.1093/sysbio/syq010	journal	March 2010
DNase H Activity of Neisseria meningitidis Cas9 Zhang, Yan; Rajan, Rakhi; Seifert, H. Steven Molecular Cell, Vol. 60, Issue 2 https://doi.org/10.1016/j.molcel.2015.09.020	journal	October 2015
Crystal Structure of Staphylococcus aureus Cas9 Nishimasu, Hiroshi; Cong, Le; Yan, Winston X. Cell, Vol. 162, Issue 5 https://doi.org/10.1016/j.cell.2015.08.007	journal	August 2015
DNA interrogation by the CRISPR RNA-guided endonuclease Cas9 Sternberg, Samuel H.; Redding, Sy; Jinek, Martin Nature, Vol. 507, Issue 7490 https://doi.org/10.1038/nature13011	journal	January 2014
Elimination of Toxic Microsatellite Repeat Expansion RNA by RNA-Targeting Cas9 Batra, Ranjan; Nelles, David A.; Pirie, Elaine Cell, Vol. 170, Issue 5 https://doi.org/10.1016/j.cell.2017.07.010	journal	August 2017
Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition Kleinstiver, Benjamin P.; Prew, Michelle S.; Tsai, Shengdar Q. Nature Biotechnology, Vol. 33, Issue 12 https://doi.org/10.1038/nbt.3404	journal	November 2015
Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch Oakes, Benjamin L.; Nadler, Dana C.; Flamholz, Avi Nature Biotechnology, Vol. 34, Issue 6 https://doi.org/10.1038/nbt.3528	journal	May 2016
PANDAseq: paired-end assembler for illumina sequences Masella, Andre P.; Bartram, Andrea K.; Truszkowski, Jakub M. BMC Bioinformatics, Vol. 13, Issue 1 https://doi.org/10.1186/1471-2105-13-31	journal	January 2012
Direct Cytosolic Delivery of CRISPR/Cas9-Ribonucleoprotein for Efficient Gene Editing Mout, Rubul; Ray, Moumita; Yesilbag Tonga, Gulen ACS Nano, Vol. 11, Issue 3 https://doi.org/10.1021/acsnano.6b07600	journal	January 2017
Mfold web server for nucleic acid folding and hybridization prediction Zuker, M. Nucleic Acids Research, Vol. 31, Issue 13 https://doi.org/10.1093/nar/gkg595	journal	July 2003
RNA-based recognition and targeting: sowing the seeds of specificity Gorski, Stanislaw A.; Vogel, Jörg; Doudna, Jennifer A. Nature Reviews Molecular Cell Biology, Vol. 18, Issue 4 https://doi.org/10.1038/nrm.2016.174	journal	February 2017
Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation Gilbert, Luke A.; Horlbeck, Max A.; Adamson, Britt Cell, Vol. 159, Issue 3 https://doi.org/10.1016/j.cell.2014.09.029	journal	October 2014
Classification and evolution of type II CRISPR-Cas systems Chylinski, Krzysztof; Makarova, Kira S.; Charpentier, Emmanuelle Nucleic Acids Research, Vol. 42, Issue 10 https://doi.org/10.1093/nar/gku241	journal	April 2014
Structure and Engineering of Francisella novicida Cas9 Hirano, Hisato; Gootenberg, Jonathan S.; Horii, Takuro Cell, Vol. 164, Issue 5 https://doi.org/10.1016/j.cell.2016.01.039	journal	February 2016
Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease Anders, Carolin; Niewoehner, Ole; Duerst, Alessia Nature, Vol. 513, Issue 7519 https://doi.org/10.1038/nature13579	journal	July 2014
UMI-tools: modeling sequencing errors in Unique Molecular Identifiers to improve quantification accuracy Smith, Tom; Heger, Andreas; Sudbery, Ian Genome Research, Vol. 27, Issue 3 https://doi.org/10.1101/gr.209601.116	journal	January 2017
A CRISPR/Cas system mediates bacterial innate immune evasion and virulence Sampson, Timothy R.; Saroj, Sunil D.; Llewellyn, Anna C. Nature, Vol. 497, Issue 7448 https://doi.org/10.1038/nature12048	journal	April 2013
C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector Abudayyeh, Omar O.; Gootenberg, Jonathan S.; Konermann, Silvana Science, Vol. 353, Issue 6299 https://doi.org/10.1126/science.aaf5573	journal	June 2016
Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems Shmakov, Sergey; Abudayyeh, Omar O.; Makarova, Kira S. Molecular Cell, Vol. 60, Issue 3, p. 385-397 https://doi.org/10.1016/j.molcel.2015.10.008	journal	November 2015
In situ structures of the genome and genome-delivery apparatus in a single-stranded RNA virus Dai, Xinghong; Li, Zhihai; Lai, Mason Nature, Vol. 541, Issue 7635 https://doi.org/10.1038/nature20589	journal	December 2016
Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications Friedland, Ari E.; Baral, Reshica; Singhal, Pankhuri Genome Biology, Vol. 16, Issue 1 https://doi.org/10.1186/s13059-015-0817-8	journal	November 2015
Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems Mohanraju, Prarthana; Makarova, Kira S.; Zetsche, Bernd Science, Vol. 353, Issue 6299 https://doi.org/10.1126/science.aad5147	journal	August 2016
DNA targeting specificity of RNA-guided Cas9 nucleases Hsu, Patrick D.; Scott, David A.; Weinstein, Joshua A. Nature Biotechnology, Vol. 31, Issue 9, p. 827-832 https://doi.org/10.1038/nbt.2647	journal	July 2013
WebLogo: A Sequence Logo Generator Crooks, Gavin E.; Hon, Gary; Chandonia, John-Marc Genome Research, Vol. 14, Issue 6, p. 1188-1190 https://doi.org/10.1101/gr.849004	journal	May 2004
Improving CRISPR-Cas nuclease specificity using truncated guide RNAs Fu, Yanfang; Sander, Jeffry D.; Reyon, Deepak Nature Biotechnology, Vol. 32, Issue 3 https://doi.org/10.1038/nbt.2808	journal	January 2014
The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA Garneau, Josiane E.; Dupuis, Marie-Ève; Villion, Manuela Nature, Vol. 468, Issue 7320 https://doi.org/10.1038/nature09523	journal	November 2010
Applications of Cas9 as an RNA-programmed RNA-binding protein Nelles, David A.; Fang, Mark Y.; Aigner, Stefan BioEssays, Vol. 37, Issue 7 https://doi.org/10.1002/bies.201500001	journal	April 2015
Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria Gasiunas, G.; Barrangou, R.; Horvath, P. Proceedings of the National Academy of Sciences, Vol. 109, Issue 39, p. E2579-E2586 https://doi.org/10.1073/pnas.1208507109	journal	September 2012
Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA Nishimasu, Hiroshi; Ran, F. Ann; Hsu, Patrick D. Cell, Vol. 156, Issue 5 https://doi.org/10.1016/j.cell.2014.02.001	journal	February 2014
Phage Response to CRISPR-Encoded Resistance in Streptococcus thermophilus Deveau, H.; Barrangou, R.; Garneau, J. E. Journal of Bacteriology, Vol. 190, Issue 4 https://doi.org/10.1128/JB.01412-07	journal	December 2007
A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity Jinek, M.; Chylinski, K.; Fonfara, I. Science, Vol. 337, Issue 6096, p. 816-821 https://doi.org/10.1126/science.1225829	journal	June 2012
edgeR: a Bioconductor package for differential expression analysis of digital gene expression data Robinson, M. D.; McCarthy, D. J.; Smyth, G. K. Bioinformatics, Vol. 26, Issue 1 https://doi.org/10.1093/bioinformatics/btp616	journal	November 2009
HTSeq--a Python framework to work with high-throughput sequencing data Anders, S.; Pyl, P. T.; Huber, W. Bioinformatics, Vol. 31, Issue 2 https://doi.org/10.1093/bioinformatics/btu638	journal	September 2014
Distinct patterns of Cas9 mismatch tolerance in vitro and in vivo Fu, Becky X. H.; St. Onge, Robert P.; Fire, Andrew Z. Nucleic Acids Research, Vol. 44, Issue 11 https://doi.org/10.1093/nar/gkw417	journal	May 2016
Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection East-Seletsky, Alexandra; O’Connell, Mitchell R.; Knight, Spencer C. Nature, Vol. 538, Issue 7624 https://doi.org/10.1038/nature19802	journal	September 2016
Programmable RNA Tracking in Live Cells with CRISPR/Cas9 Nelles, David A.; Fang, Mark Y.; O’Connell, Mitchell R. Cell, Vol. 165, Issue 2 https://doi.org/10.1016/j.cell.2016.02.054	journal	April 2016
Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation McCarthy, Davis J.; Chen, Yunshun; Smyth, Gordon K. Nucleic Acids Research, Vol. 40, Issue 10 https://doi.org/10.1093/nar/gks042	journal	January 2012
PROMALS3D: a tool for multiple protein sequence and structure alignments Pei, Jimin; Kim, Bong-Hyun; Grishin, Nick V. Nucleic Acids Research, Vol. 36, Issue 7 https://doi.org/10.1093/nar/gkn072	journal	February 2008
Fast gapped-read alignment with Bowtie 2 Langmead, Ben; Salzberg, Steven L. Nature Methods, Vol. 9, Issue 4 https://doi.org/10.1038/nmeth.1923	journal	March 2012
Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes Szczelkun, Mark D.; Tikhomirova, Maria S.; Sinkunas, Tomas Proceedings of the National Academy of Sciences, Vol. 111, Issue 27 https://doi.org/10.1073/pnas.1402597111	journal	May 2014
Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28 Smargon, Aaron A.; Cox, David B. T.; Pyzocha, Neena K. Molecular Cell, Vol. 65, Issue 4 https://doi.org/10.1016/j.molcel.2016.12.023	journal	February 2017
Mechanism of substrate selection by a highly specific CRISPR endoribonuclease Sternberg, S. H.; Haurwitz, R. E.; Doudna, J. A. RNA, Vol. 18, Issue 4 https://doi.org/10.1261/rna.030882.111	journal	February 2012
Forna (force-directed RNA): Simple and effective online RNA secondary structure diagrams Kerpedjiev, Peter; Hammer, Stefan; Hofacker, Ivo L. Bioinformatics, Vol. 31, Issue 20 https://doi.org/10.1093/bioinformatics/btv372	journal	June 2015
Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo Zuris, John A.; Thompson, David B.; Shu, Yilai Nature Biotechnology, Vol. 33, Issue 1 https://doi.org/10.1038/nbt.3081	journal	October 2014
Fast and effective prediction of microRNA/target duplexes Rehmsmeier, M. RNA, Vol. 10, Issue 10 https://doi.org/10.1261/rna.5248604	journal	October 2004
A Cas9-guide RNA complex preorganized for target DNA recognition Jiang, F.; Zhou, K.; Ma, L. Science, Vol. 348, Issue 6242 https://doi.org/10.1126/science.aab1452	journal	June 2015
Multiplex Genome Engineering Using CRISPR/Cas Systems Cong, L.; Ran, F. A.; Cox, D. Science, Vol. 339, Issue 6121, p. 819-823 https://doi.org/10.1126/science.1231143	journal	January 2013
Molecular structure of r(GCG)d(TATACGC): a DNA–RNA hybrid helix joined to double helical DNA Wang, Andrew H. -J.; Fujii, Satoshi; van Boom, Jacques H. Nature, Vol. 299, Issue 5884 https://doi.org/10.1038/299601a0	journal	October 1982
Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems Yamada, Mari; Watanabe, Yuto; Gootenberg, Jonathan S. Molecular Cell, Vol. 65, Issue 6 https://doi.org/10.1016/j.molcel.2017.02.007	journal	March 2017
Lessons from Enzyme Kinetics Reveal Specificity Principles for RNA-Guided Nucleases in RNA Interference and CRISPR-Based Genome Editing Bisaria, Namita; Jarmoskaite, Inga; Herschlag, Daniel Cell Systems, Vol. 4, Issue 1 https://doi.org/10.1016/j.cels.2016.12.010	journal	January 2017
In vivo genome editing using Staphylococcus aureus Cas9 Ran, F. Ann; Cong, Le; Yan, Winston X. Nature, Vol. 520, Issue 7546 https://doi.org/10.1038/nature14299	journal	April 2015
In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni Kim, Eunji; Koo, Taeyoung; Park, Sung Wook Nature Communications, Vol. 8, Issue 1 https://doi.org/10.1038/ncomms14500	journal	February 2017
RNA-guided editing of bacterial genomes using CRISPR-Cas systems Jiang, Wenyan; Bikard, David; Cox, David Nature Biotechnology, Vol. 31, Issue 3, p. 233-239 https://doi.org/10.1038/nbt.2508	journal	January 2013
SHAPE-Seq 2.0: systematic optimization and extension of high-throughput chemical probing of RNA secondary structure with next generation sequencing Loughrey, David; Watters, Kyle E.; Settle, Alexander H. Nucleic Acids Research, Vol. 42, Issue 21 https://doi.org/10.1093/nar/gku909	journal	October 2014
Filter-Binding Assay for Analysis of RNA-Protein Interactions Rio, D. C. Cold Spring Harbor Protocols, Vol. 2012, Issue 10 https://doi.org/10.1101/pdb.prot071449	journal	October 2012
Targeting cellular mRNAs translation by CRISPR-Cas9 Liu, Yuchen; Chen, Zhicong; He, Anbang Scientific Reports, Vol. 6, Issue 1 https://doi.org/10.1038/srep29652	journal	July 2016

Similar Records

RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System

Journal Article · Mon Jan 23 00:00:00 EST 2017 · PLoS ONE · OSTI ID:1415873

Liu, Tina Y.; Iavarone, Anthony T.; Doudna, Jennifer A.

Protospacer Adjacent Motif-Induced Allostery Activates CRISPR-Cas9

Journal Article · Wed Aug 02 00:00:00 EDT 2017 · Journal of the American Chemical Society · OSTI ID:1415873

Palermo, Giulia; Ricci, Clarisse G.; Fernando, Amendra; +5 more

Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9

Journal Article · Wed Sep 14 00:00:00 EDT 2016 · Nature Communications · OSTI ID:1415873

Singh, Digvijay; Sternberg, Samuel H.; Fei, Jingyi; +2 more

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY

Title: RNA-dependent RNA targeting by CRISPR-Cas9

Citation Formats

References (65)

Similar Records

Related Subjects