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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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.
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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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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