Synthetic CRISPR RNA-Cas9–guided genome editing in human cells

Significance Genome editing with nucleases that recognize specific DNA sequences is a powerful technology for manipulating genomes. This is especially true for the Cas9 nuclease, the site specificity of which is determined by a bound RNA, called a CRISPR RNA (crRNA). Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA) and show that it can functionally replace the natural crRNA, producing enhanced cleavage activity at a target DNA site with apparently reduced off-target cleavage. scrRNAs can be synthesized in a commercially feasible manner today and provide a platform for therapeutic applications. Genome editing with the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nuclease system is a powerful technology for manipulating genomes, including introduction of gene disruptions or corrections. Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA), which in combination with unmodified transactivating crRNA (tracrRNA) is shown to functionally replace the natural guide RNA in the CRISPR-Cas9 nuclease system and to mediate efficient genome editing in human cells. Incorporation of rational chemical modifications known to protect against nuclease digestion and stabilize RNA–RNA interactions in the tracrRNA hybridization region of CRISPR RNA (crRNA) yields a scrRNA with enhanced activity compared with the unmodified crRNA and comparable gene disruption activity to the previously published single guide RNA. Taken together, these findings provide a platform for therapeutic applications, especially for nervous system disease, using successive application of cell-permeable, synthetic CRISPR RNAs to activate and then silence Cas9 nuclease activity.