Chemically Synthesized Modified Guide RNAs Enhance CRISPR/Cas Genome Editing

Using recent advancements in RNA synthesis technology (Dellinger et al. J. Am. Chem. Soc. 2011, 133, 11540–11556), we find it straightforward to chemically synthesize RNAs of more than 100 nucleotides in length and to incorporate modified nucleotides at any position. Using this technology, we recently demonstrated 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 (Hendel et al. Nat. Biotech. 2015 doi:10.1038/nbt.3290). When co-delivering chemically modified sgRNAs and Cas9 mRNA we observe >90% in/del frequencies in human cells lines, ~70% in human primary T cells, and ~40% in CD34+ hematopoietic stem and progenitor cells (HSPCs), despite detecting no activity for unmodified sgRNAs in the latter two cell types.  We observe no apparent cellular toxicity and analysis of off-target activities indicate that chemically modified sgRNAs typically retain high specificity.  Here, we extend this work by characterizing the performance of chemically modified sgRNAs for genome editing in additional cell types including human immortalized, primary and induced pluripotent stem cells. The ability to chemically synthesize high quality sgRNAs in a scalable manner enables their application in all phases from basic research to targeted therapies.