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

Ayal Hendel; Rasmus O. Bak; Joseph T. Clark; Andrew B. Kennedy; Daniel E. Ryan; Subhadeep Roy; Israel Steinfeld; Benjamin D. Lunstad; Robert J. Kaiser; Alec B. Wilkens; Rosa Bacchetta; Anya Tsalenko; Douglas Dellinger; Laurakay Bruhn; Matthew H. Porteus

doi:10.1038/nbt.3290

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

Ayal Hendel, Rasmus O. Bak, Joseph T. Clark, Andrew B. Kennedy, Daniel E. Ryan, Subhadeep Roy, Israel Steinfeld, Benjamin D. Lunstad, Robert J. Kaiser, Alec B. Wilkens, Rosa Bacchetta, Anya Tsalenko, Douglas Dellinger, Laurakay Bruhn, Matthew H. Porteus

Research output: Contribution to journal › Article › peer-review

880 Scopus citations

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.

Original language	English
Pages (from-to)	985-989
Number of pages	5
Journal	Nature Biotechnology
Volume	33
Issue number	9
DOIs	https://doi.org/10.1038/nbt.3290
State	Published - 10 Sep 2015
Externally published	Yes

Bibliographical note

Publisher Copyright:
© 2015 Nature America, Inc. All rights reserved.

Funding

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.

Funders	Funder number
Medical Sciences
National Institutes of Health	PN2EY018244
National Institute of Allergy and Infectious Diseases	R01AI097320
Amon G. Carter Foundation
Myotonic Dystrophy Foundation	DFF–1331-00735B, DFF–1333-00106B
Danmarks Frie Forskningsfond

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Hendel, A., Bak, R. O., Clark, J. T., Kennedy, A. B., Ryan, D. E., Roy, S., Steinfeld, I., Lunstad, B. D., Kaiser, R. J., Wilkens, A. B., Bacchetta, R., Tsalenko, A., Dellinger, D., Bruhn, L., & Porteus, M. H. (2015). Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nature Biotechnology, 33(9), 985-989. https://doi.org/10.1038/nbt.3290

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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.",

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

Abstract

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