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

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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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).

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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.

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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.

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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.

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

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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

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