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:

Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells

Nature Biotechnology volume 33, pages 985–989 (2015)Cite this article

Subjects

Abstract

CRISPR-Cas-mediated genome editing relies on guide RNAs that direct site-specific DNA cleavage facilitated by the Cas endonuclease. Here we report that chemical alterations to synthesized single guide RNAs (sgRNAs) enhance genome editing efficiency in human primary T cells and CD34+ hematopoietic stem and progenitor cells. Co-delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system, without the toxicity associated with DNA delivery. This approach is a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Synthesized and chemically modified sgRNAs facilitate high frequencies of indels and HR in the human cell line K562.
Figure 2: Chemically modified sgRNAs facilitate high frequencies of gene disruption in stimulated primary human T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs).

Similar content being viewed by others

References

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Cong, L. et al. Multiplex genome engineering using CRISPR-Cas systems. Science 339, 819–823 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Hsu, P.D., Lander, E.S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hendel, A., Fine, E.J., Bao, G. & Porteus, M.H. Quantifying on- and off-target genome editing. Trends Biotechnol. 33, 132–140 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Dellinger, D.J. et al. Streamlined process for the chemical synthesis of RNA using 2′-O-thionocarbamate-protected nucleoside phosphoramidites in the solid phase. J. Am. Chem. Soc. 133, 11540–11556 (2011).

    Article  CAS  PubMed  Google Scholar 

  7. Deleavey, G.F. & Damha, M.J. Designing chemically modified oligonucleotides for targeted gene silencing. Chem. Biol. 19, 937–954 (2012).

    Article  CAS  PubMed  Google Scholar 

  8. Eckstein, F. Phosphorothioates, essential components of therapeutic oligonucleotides. Nucleic Acid Ther. 24, 374–387 (2014).

    Article  CAS  PubMed  Google Scholar 

  9. Dellinger, D.J., Sheehan, D.M., Christensen, N.K., Lindberg, J.G. & Caruthers, M.H. Solid-phase chemical synthesis of phosphonoacetate and thiophosphonoacetate oligodeoxynucleotides. J. Am. Chem. Soc. 125, 940–950 (2003).

    Article  CAS  PubMed  Google Scholar 

  10. Hendel, A. et al. Quantifying genome-editing outcomes at endogenous loci with SMRT sequencing. Cell Reports 7, 293–305 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Cradick, T.J., Fine, E.J., Antico, C.J. & Bao, G. CRISPR-Cas9 systems targeting beta-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res. 41, 9584–9592 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Tebas, P. et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N. Engl. J. Med. 370, 901–910 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Lombardo, A. et al. Site-specific integration and tailoring of cassette design for sustainable gene transfer. Nat. Methods 8, 861–869 (2011).

    Article  CAS  PubMed  Google Scholar 

  14. Voit, R.A., Hendel, A., Pruett-Miller, S.M. & Porteus, M.H. Nuclease-mediated gene editing by homologous recombination of the human globin locus. Nucleic Acids Res. 42, 1365–1378 (2014).

    Article  CAS  PubMed  Google Scholar 

  15. Hsu, P.D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Cradick, T.J., Qiu, P., Lee, C.M., Fine, E.J. & Bao, G. COSMID: a web-based tool for identifying and validating CRISPR-Cas off-target sites. Mol. Ther. Nucleic Acids 3, e214 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Yi, G. et al. CCR5 gene editing of resting CD4(+) T cells by transient ZFN expression from HIV envelope pseudotyped nonintegrating lentivirus confers HIV-1 resistance in humanized mice. Mol. Ther. Nucleic Acids 3, e198 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Mandal, P.K. et al. Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR-Cas9. Cell Stem Cell 15, 643–652 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Gilbert, L.A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 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  PubMed  PubMed Central  Google Scholar 

  21. O'Connell, M.R. et al. Programmable RNA recognition and cleavage by CRISPR-Cas9. Nature 516, 263–266 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Threlfall, R.N., Torres, A.G., Krivenko, A., Gait, M.J. & Caruthers, M.H. Synthesis and biological activity of phosphonoacetate- and thiophosphonoacetate-modified 2′-O-methyl oligoribonucleotides. Org. Biomol. Chem. 10, 746–754 (2012).

    Article  CAS  PubMed  Google Scholar 

  23. Brinkman, E.K., Chen, T., Amendola, M. & van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, e168 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589–595 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

A.H. was supported by the Myotonic Dystrophy Foundation. R.O.B. was supported through an Individual Postdoctoral grant (DFF–1333-00106B) and a Sapere Aude, Research Talent grant (DFF–1331-00735B), both from the Danish Council for Independent Research, Medical Sciences. M.H.P. gratefully acknowledges the support of the Amon Carter Foundation, the Laurie Krauss Lacob Faculty Scholar Award in Pediatric Translational Research and US National Institutes of Health grant support PN2EY018244 and R01-AI097320. We thank R. Perriman, and Porteus laboratory members and C. Carstens, S. Laderman and Agilent laboratories members for helpful input, comments and discussion.

Author information

Author notes

  1. Ayal Hendel and Rasmus O Bak: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pediatrics, Stanford University, Stanford, California, USA

    Ayal Hendel, Rasmus O Bak, Joseph T Clark, Alec B Wilkens, Rosa Bacchetta & Matthew H Porteus

  2. Agilent Research Laboratories, Santa Clara, California, USA

    Andrew B Kennedy, Daniel E Ryan, Robert J Kaiser, Anya Tsalenko & Laurakay Bruhn

  3. Agilent Research Laboratories, Boulder, Colorado, USA

    Subhadeep Roy, Benjamin D Lunstad & Douglas Dellinger

  4. Agilent Research Laboratories, Tel Aviv, Israel

    Israel Steinfeld

Authors
  1. Ayal Hendel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rasmus O Bak
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joseph T Clark
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andrew B Kennedy
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Daniel E Ryan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Subhadeep Roy
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Israel Steinfeld
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Benjamin D Lunstad
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Robert J Kaiser
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Alec B Wilkens
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Rosa Bacchetta
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Anya Tsalenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Douglas Dellinger
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Laurakay Bruhn
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Matthew H Porteus
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.H. and R.O.B. contributed equally to this work. A.H., R.O.B., J.T.C., A.B.W., R.B., A.B.K. and D.E.R. performed and designed experiments. D.D., D.E.R. and R.J.K. chose the specific modification types and the positions for their incorporation into the sgRNAs. S.R., B.D.L. and R.J.K. performed synthesis and purification of all the sgRNAs. I.S. and A.T. developed and applied the sequencing data analysis pipeline used to analyze the deep sequencing data. L.B. and M.H.P. directed the research. A.H. and R.O.B. wrote the manuscript with help from all authors.

Corresponding authors

Correspondence to Laurakay Bruhn or Matthew H Porteus.

Ethics declarations

Competing interests

A.B.K., D.E.R., I.S., B.D.L., R.J.K., A.T., D.D. and L.B. are employees of Agilent Technologies. M.H.P. is a consultant and has equity interest in CRISPR Tx, but CRISPR Tx had no input into the design, execution, interpretation or publication of the results herein.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–16, Supplementary Tables 1–5 and Supplementary Notes 1 and 2 (PDF 999 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hendel, A., Bak, R., Clark, J. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat Biotechnol 33, 985–989 (2015). https://doi.org/10.1038/nbt.3290

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.3290

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research