5′ modifications to CRISPR–Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage

A key aim in exploiting CRISPR–Cas is gRNA engineering to introduce additional functionalities, ranging from individual nucleotide changes that increase efficiency of on-target binding to the inclusion of larger functional RNA aptamers or ribonucleoproteins (RNPs). Cas9–gRNA interactions are crucial for complex assembly, but several distinct regions of the gRNA are amenable to modification. We used in vitro ensemble and single-molecule assays to assess the impact of gRNA structural alterations on RNP complex formation, R-loop dynamics, and endonuclease activity. Our results indicate that RNP formation was unaffected by any of our modifications. R-loop formation and DNA cleavage activity were also essentially unaffected by modification of the Upper Stem, first Hairpin and 3′ end. In contrast, we found that 5′ additions of only two or three nucleotides could reduce R-loop formation and cleavage activity of the RuvC domain relative to a single nucleotide addition. Such modifications are a common by-product of in vitro transcribed gRNA. We also observed that addition of a 20 nt RNA hairpin to the 5′ end of a gRNA still supported RNP formation but produced a stable ∼9 bp R-loop that could not activate DNA cleavage. Consideration of these observations will assist in successful gRNA design.