Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding

Abstract

Cas9 has made a wide range of genomic manipulation possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity, but at the cost of the on-target activity. DNA unwinding is a checkpoint before cleavage by Cas9, and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in engineered Cas9s. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used, but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two non-matching guanines added to the 5′ end were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5′ end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.

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

“…It may be that the effect seen here is unique to the RP hairpin and that empirical design can be used to select for RNA structures that have the desired effect and to avoid unwanted ones. There may also be protospacer specific effects, as observed for the short 5′ modifications (13). The unusual activity of 5′ RP gRNA could be exploited to form stable R-loops without activating DNA cleavage.…”

“…In their recent study, Okafor et al suggested that effects of unpaired 5′ nucleotides can be sequence context specific (13). To explore this, we targeted an alternative protospacer sequence on pSP1, where the 20th nucleotide on the protospacer DNA targeted strand is a cytosine that can base pair with a terminal 5′ guanine on the gRNA spacer ( Figure 3A).…”

“…Okafor et al (13) proposed that due to close interaction between the RuvC nuclease domain and the 5′ end of the spacer RNA, unpaired residues may alter Cas9 activity. The slower rate of second strand cleavage observed here with both the 1025 and 1166 gRNAs (Figures 2 and 3) could therefore be due to misalignment of the RuvC nuclease that slows cleavage of the non-targeted strand.…”

“…Unpaired nucleotides at the 5′ end of the spacer sequence can influence SpCas9 activity in a protein and target site-dependent manner (12,13). For WT Cas9, one or two additional 5′ guanines lead to increased specificity due to reduced unwinding promiscuity.…”

“…Other engineered Cas9s showed either reduced on-target activity (eCas9 and HypaCas9) or increased promiscuity (Cas9-HF1). These pleiotropic effects are most likely due to the observed interactions of the docked RuvC domain with the 5′ end of the RNA (9,13). Unpaired 5′ nucleotides could produce distortion of the DNA:RNA hybrid in this region that may influence R-loop formation, and possibly DNA cleavage.…”