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

  • Nat Biotechnol. 2015 Sep;33(9):985-989. doi: 10.1038/nbt.3290.
Ayal Hendel  #  1 ,  Rasmus O Bak  #  1 ,  Joseph T Clark  1 ,  Andrew B Kennedy  2 ,  Daniel E Ryan  2 ,  Subhadeep Roy  3 ,  Israel Steinfeld  4 ,  Benjamin D Lunstad  3 ,  Robert J Kaiser  2 ,  Alec B Wilkens  1 ,  Rosa Bacchetta  1 ,  Anya Tsalenko  2 ,  Douglas Dellinger  3 ,  Laurakay Bruhn  2 ,  Matthew H Porteus  1
Affiliations
  • 1. Department of Pediatrics, Stanford University, Stanford, California, USA.
  • 2. Agilent Research Laboratories, Santa Clara, California, USA.
  • 3. Agilent Research Laboratories, Boulder, Colorado, USA.
  • 4. Agilent Research Laboratories, Tel Aviv, Israel.
  • # Contributed equally.
PMID: 26121415 DOI: 10.1038/nbt.3290
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.