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.