Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Programmable RNA recognition and cleavage by CRISPR/Cas9

Abstract

The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA–DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage1,2,3,4,5. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA4,5,6,7. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms8, but it has been thought to be incapable of targeting RNA5. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage7. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: RNA-guided Cas9 cleaves ssRNA targets in the presence of a short PAM-presenting DNA oligonucleotide (PAMmer).
Figure 2: dCas9–gRNA binds ssRNA targets with high affinity in the presence of PAMmers.
Figure 3: 5′-extended PAMmers are required for specific target ssRNA binding.
Figure 4: RNA-guided Cas9 can target non-PAM sites on ssRNA and isolate GAPDH mRNA from HeLa cells in a tagless manner.

Similar content being viewed by others

References

  1. Wiedenheft, B., Sternberg, S. H. & Doudna, J. A. RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331–338 (2012)

    Article  ADS  CAS  Google Scholar 

  2. Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007)

    Article  ADS  CAS  Google Scholar 

  3. Garneau, J. E. et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67–71 (2010)

    Article  ADS  CAS  Google Scholar 

  4. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012)

    Article  ADS  CAS  Google Scholar 

  5. Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl Acad. Sci. USA 109, E2579–E2586 (2012)

    Article  ADS  CAS  Google Scholar 

  6. Mojica, F. J. M., Diez-Villasenor, C., Garcia-Martinez, J. & Almendros, C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiol 155, 733–740 (2009)

    Article  CAS  Google Scholar 

  7. Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C. & Doudna, J. A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62–67 (2014)

    Article  ADS  CAS  Google Scholar 

  8. Mali, P., Esvelt, K. M. & Church, G. M. Cas9 as a versatile tool for engineering biology. Nature Methods 10, 957–963 (2013)

    Article  CAS  Google Scholar 

  9. Marraffini, L. A. & Sontheimer, E. J. Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature 463, 568–571 (2010)

    Article  ADS  CAS  Google Scholar 

  10. Sashital, D. G., Wiedenheft, B. & Doudna, J. A. Mechanism of foreign DNA selection in a bacterial adaptive immune system. Mol. Cell 46, 606–615 (2012)

    Article  CAS  Google Scholar 

  11. Pommer, A. J. et al. Mechanism and cleavage specificity of the H-N-H endonuclease colicin E9. J. Mol. Biol. 314, 735–749 (2001)

    Article  CAS  Google Scholar 

  12. Hsia, K. C. et al. DNA binding and degradation by the HNH protein ColE7. Structure 12, 205–214 (2004)

    Article  Google Scholar 

  13. Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014)

    Article  CAS  Google Scholar 

  14. Wu, H. J., Lima, W. F. & Crooke, S. T. Properties of cloned and expressed human RNase H1. J. Biol. Chem. 274, 28270–28278 (1999)

    Article  CAS  Google Scholar 

  15. Engreitz, J. M. et al. The Xist lncRNA exploits three-dimensional genome architecture to spread across the X chromosome. Science 341, (2013)

  16. Chu, C., Qu, K., Zhong, F. L., Artandi, S. E. & Chang, H. Y. Genomic maps of long noncoding RNA occupancy reveal principles of RNA–chromatin interactions. Mol. Cell 44, 667–678 (2011)

    Article  CAS  Google Scholar 

  17. Simon, M. D. et al. The genomic binding sites of a noncoding RNA. Proc. Natl Acad. Sci. USA 108, 20497–20502 (2011)

    Article  ADS  CAS  Google Scholar 

  18. Mackay, J. P., Font, J. & Segal, D. J. The prospects for designer single-stranded RNA-binding proteins. Nature Struct. Mol. Biol. 18, 256–261 (2011)

    Article  CAS  Google Scholar 

  19. Filipovska, A. & Rackham, O. Designer RNA-binding proteins: new tools for manipulating the transcriptome. RNA Biol. 8, 978–983 (2011)

    Article  CAS  Google Scholar 

  20. Wang, Y., Wang, Z. & Tanaka Hall, T. M. Engineered proteins with Pumilio/fem-3 mRNA binding factor scaffold to manipulate RNA metabolism. FEBS J. 280, 3755–3767 (2013)

    Article  CAS  Google Scholar 

  21. Yin, P. et al. Structural basis for the modular recognition of single-stranded RNA by PPR proteins. Nature 504, 168–171 (2013)

    Article  ADS  CAS  Google Scholar 

  22. Yagi, Y., Nakamura, T. & Small, I. The potential for manipulating RNA with pentatricopeptide repeat proteins. Plant J. 78, 772–782 (2014)

    Article  CAS  Google Scholar 

  23. Kim, D. H. & Rossi, J. J. RNAi mechanisms and applications. Biotechniques 44, 613–616 (2008)

    Article  CAS  Google Scholar 

  24. Hale, C. R. et al. RNA-guided RNA cleavage by a CRISPR RNA–Cas protein complex. Cell 139, 945–956 (2009)

    Article  CAS  Google Scholar 

  25. Hale, C. R. et al. Essential features and rational design of CRISPR RNAs that function with the Cas RAMP module complex to cleave RNAs. Mol. Cell 45, 292–302 (2012)

    Article  CAS  Google Scholar 

  26. Staals, R. H. J. et al. Structure and activity of the RNA-targeting type III-B CRISPR-Cas complex of Thermus thermophilus. Mol. Cell 52, 135–145 (2013)

    Article  CAS  Google Scholar 

  27. Spilman, M. et al. Structure of an RNA silencing complex of the CRISPR-Cas immune system. Mol. Cell 52, 146–152 (2013)

    Article  ADS  CAS  Google Scholar 

  28. Terns, R. M. & Terns, M. P. CRISPR-based technologies: prokaryotic defense weapons repurposed. Trends Genet. 30, 111–118 (2014)

    Article  CAS  Google Scholar 

  29. Sampson, T. R., Saroj, S. D., Llewellyn, A. C., Tzeng, Y. L. & Weiss, D. S. A. CRISPR/Cas system mediates bacterial innate immune evasion and virulence. Nature 497, 254–257 (2013)

    Article  ADS  CAS  Google Scholar 

  30. Sampson, T. R. & Weiss, D. S. Exploiting CRISPR/Cas systems for biotechnology. Bioessays 36, 34–38 (2014)

    Article  CAS  Google Scholar 

  31. Sternberg, S. H., Haurwitz, R. E. & Doudna, J. A. Mechanism of substrate selection by a highly specific CRISPR endoribonuclease. RNA 18, 661–672 (2012)

    Article  CAS  Google Scholar 

  32. Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491 (2013)

    Article  CAS  Google Scholar 

  33. Lee, H. Y. et al. RNA-protein analysis using a conditional CRISPR nuclease. Proc. Natl Acad. Sci. USA 110, 5416–5421 (2013)

    Article  ADS  CAS  Google Scholar 

  34. Chomczynski, P. One-hour downward alkaline capillary transfer for blotting of DNA and RNA. Anal. Biochem. 201, 134–139 (1992)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank B. Staahl and K. Zhou for technical assistance, A. Iavarone for assistance with mass spectrometry measurements, Integrated DNA Technologies for the synthesis of DNA and RNA oligonucleotides, and members of the Doudna laboratory and J. Cate for discussions and critical reading of the manuscript. S.H.S. acknowledges support from the National Science Foundation and National Defense Science & Engineering Graduate Research Fellowship programs. A.E.-S. and B.L.O. acknowledge support from NIH NRSA trainee grants. Funding was provided by the NIH-funded Center for RNA Systems Biology (P50GM102706-03). J.A.D. is an Investigator of the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

Authors

Contributions

M.R.O. and S.H.S. conceived the project. M.R.O., B.L.O., S.H.S., A.E.-S. and M.K. conducted experiments. All authors discussed the data, and M.R.O., S.H.S., B.L.O. and J.A.D. wrote the manuscript.

Corresponding author

Correspondence to Jennifer A. Doudna.

Ethics declarations

Competing interests

J.A.D., M.R.O., B.L.O. and S.H.S. are inventors on a related patent application.

Extended data figures and tables

Extended Data Figure 1 Quantified data for cleavage of ssRNA by Cas9–gRNA in the presence of a 19-nucleotide PAMmer.

Cleavage assays were conducted as described in the Methods, and the quantified data were fitted with single-exponential decays. Results from four independent experiments yielded an average apparent pseudo-first-order cleavage rate constant (mean ± s.d.) of 0.032 ± 0.007 min−1. This is slower than the rate constant determined previously for ssDNA in the presence of the same 19-nucleotide PAMmer (7.3 ± 3.2 min−1)7.

Extended Data Figure 2 RNA cleavage is marginally stimulated by di- and tri-deoxyribonucleotide PAMmers.

Cleavage reactions contained 1 nM 5′-radiolabelled target ssRNA and no PAMmer (left), 100 nM 18-nt PAMmer (second from left), or 1 mM of the indicated di- or tri-nucleotide (remaining lanes). Reaction products were resolved by 12% denaturing polyacrylamide gel electrophoresis (PAGE) and visualized by phosphorimaging.

Extended Data Figure 3 Representative binding experiment demonstrating guide-specific ssRNA binding with 5′-extended PAMmers.

Gel shift assays were conducted as described in the Methods. Binding reactions contained Cas9 programmed with λ2 gRNA and either λ2 (on-target), λ3 (off-target) or λ4 (off-target) ssRNA in the presence of short cognate PAMmers or cognate PAMmers with complete 5′-extensions, as indicated. The presence of a cognate 5′-extended PAM-mer abrogates off-target binding. Three independent experiments were conducted to produce the data shown in Fig. 3b, d.

Extended Data Figure 4 Exploration of RNA cleavage efficiencies and binding specificity using PAMmers with variable 5′-extensions.

a, Cleavage assays were conducted as described in Methods. Reactions contained Cas9 programmed with λ2 gRNA and λ2 ssRNA targets in the presence of PAMmers with 5′-extensions of variable length. The ssRNA cleavage efficiency decreases as the PAMmer extends further into the target region, as indicated by the fraction of RNA cleaved after 1 h. b, Binding assays were conducted as described in the Methods, using mostly the same panel of 5′-extended PAMmers as in a. Binding reactions contained Cas9 programmed with λ2 gRNA and either λ2 (on-target) or λ3 (off-target) ssRNA in the presence of cognate PAMmers with 5′-extensions of variable length. The binding specificity increases as the PAMmer extends further into the target region, as indicated by the fraction of λ3 (off-target) ssRNA bound at 3 nM Cas9–gRNA. PAMmers with 5′ extensions also cause a slight reduction in the relative binding affinity of λ2 (on-target) ssRNA.

Extended Data Figure 5 Site-specific biotin labelling of Cas9.

a, In order to introduce a single biotin moiety on Cas9, the solvent accessible, non-conserved amino-terminal methionine was mutated to a cysteine (M1C; red text) and the naturally occurring cysteine residues were mutated to serine (C80S and C574S; bold text). This enabled cysteine-specific labelling with EZ-link Maleimide-PEG2-biotin through an irreversible reaction between the reduced sulphydryl group of the cysteine and the maleimide group present on the biotin label. Mutations of dCas9 are also indicated in the domain schematic. b, Mass spectrometry analysis of the Cas9 biotin-labelling reaction confirmed that successful biotin labelling only occurs when the M1C mutation is present in the Cys-free background (C80S;C574S). The mass of the Maleimide-PEG2-biotin reagent is 525.6 Da. c, Streptavidin bead binding assay with biotinylated (biot.) or non-biotinylated (non-biot.) Cas9 and streptavidin agarose or streptavidin magnetic beads. Cas9 only remains specifically bound to the beads after biotin labelling. d, Cleavage assays were conducted as described in the Methods and resolved by denaturing PAGE. Reactions contained 100 nM Cas9 programmed with λ2 gRNA and 1 nM 5′-radiolabelled λ2 dsDNA target. e, Quantified cleavage data from triplicate experiments were fitted with single-exponential decays to calculate the apparent pseudo-first-order cleavage rate constants (average ± standard deviation). Both Cys-free and biotin-labelled Cas9(M1C) retain wild-type activity.

Extended Data Figure 6 RNA-guided Cas9 can utilize chemically modified PAMmers.

Nineteen-nucleotide PAMmer derivatives containing various chemical modifications on the 5′ and 3′ ends (capped) or interspersed throughout the strand still activate Cas9 for cleavage of ssRNA targets. These types of modification are often used to increase the in vivo half-life of short oligonucleotides by preventing exo- and endonuclease-mediated degradation. Cleavage assays were conducted as described in the Methods. PS, phosphorothioate bonds; LNA, locked nucleic acid.

Extended Data Figure 7 Cas9 programmed with GAPDH-specific gRNAs can pull down GAPDH mRNA in the absence of PAMmers.

a, Northern blot showing that, in some cases, Cas9–gRNA is able to pull down detectable amounts of GAPDH mRNA from total RNA without requiring a PAMmer. b, Northern blot showing that Cas9–gRNA G1 is also able to pull down quantitative amounts of GAPDH mRNA from HeLa cell lysate without requiring a PAMmer. s, standard; v1-5, increasingly 2′-OMe-modified PAMmers. See Fig. 4g for PAMmer sequences.

Extended Data Figure 8 Potential applications of RCas9 for untagged transcript analysis, detection and manipulation.

a, Catalytically active RCas9 could be used to target and cleave RNA, particularly those for which RNA-interference-mediated repression/degradation is not possible. b, Tethering the eukaryotic initiation factor eIF4G to a catalytically inactive dRCas9 targeted to the 5′ untranslated region of an mRNA could drive translation. c, dRCas9 tethered to beads could be used to specifically isolate RNA or native RNA–protein complexes of interest from cells for downstream analysis or assays including identification of bound-protein complexes, probing of RNA structure under native protein-bound conditions, and enrichment of rare transcripts for sequencing analysis. d, dRCas9 tethered to RNA deaminase or N6-mA methylase domains could direct site-specific A-to-I editing or methylation of RNA, respectively. e, dRCas9 fused to a U1 recruitment domain (arginine- and serine-rich (RS) domain) could be programmed to recognize a splicing enhancer site and thereby promote the inclusion of a targeted exon. f, dRCas9 tethered to a fluorescent protein such as GFP could be used to observe RNA localization and transport in living cells. Adapted from Mackay et al.18

Extended Data Table 1 λ-Oligonucleotide sequences
Extended Data Table 2 Oligonucleotides used in the GAPDH mRNA pull-down experiment

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

O’Connell, M., Oakes, B., Sternberg, S. et al. Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature 516, 263–266 (2014). https://doi.org/10.1038/nature13769

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature13769

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing