Cas9-mediated DNA cleavage guided by enzymatically prepared 4′-thio-modified RNA

CRISPR-Cas9-mediated DNA editing relies on guide RNAs (gRNAs) that direct site-specific DNA cleavage by the Cas endonuclease. Because natural gRNA is susceptible to intracellular degradation, it is desirable to chemically protect it for efficient editing. Using 4′-thioribonucleoside 5′-triphosphates and T7 transcription, we have prepared 4′-thio-modified gRNAs that guide Cas9-mediated DNA cleavage. This approach is a simple way to obtain chemically modified RNA suitable for CRISPR-Cas9 DNA editing. The 4′-thio sgRNA, which is enzymatically prepared by T7 transcription using 4′-thiopyrimidine nucleoside 5′- triphosphates, induces Cas9-mediated DNA cleavage.