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 genome engineering applications 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 the primary checkpoint before cleavage by Cas9 and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in Cas9. 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 guanines added 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.

BEAR reveals that increased fidelity variants can successfully reduce the mismatch-tolerance of adenine but not cytosine base editors

“…An interesting nding from our experiments is the low activity of the increased delity variant HeF-ABE, but not HeF-CBE. Fidelity-increasing mutations have been shown to be associated with somewhat reduced nuclease activity: these nuclease variants are reported to pass through the quality checkpoints less e ciently 43,52 than the wild type SpCas9 53, 54, 55 during target cleavage, while their binding to the target DNA is largely unaffected 43,45 . This may be manifested in lower cleavage activity on off-targets when there are mismatches between the spacer and the target DNA sequences, and sometimes on ontarget sequences as well 39,41,42,43,44,45,56 .…”

“…This may be manifested in lower cleavage activity on off-targets when there are mismatches between the spacer and the target DNA sequences, and sometimes on ontarget sequences as well 39,41,42,43,44,45,56 . With such non-cleavable targets, the variants separate the DNA strands in the PAM-proximal region upon binding to the target, however, the mutations inhibit the effective full-length separation of the two strands of the target DNA, and thus they also prevent the formation of the cleavage-competent conformation of SpCas9 52,55,57 . HeF-SpCas9, which has the highest delity and lowest average cleavage activity among these variants, binds to the targets of WT-SpCas9 without being able to cleave most of them: in contrast to the wild type nuclease 53,54,55 , it fails to effectively separate the strands of the target DNA at full length and to acquire the cleavage-competent conformation 43,45,52 .…”

“…With such non-cleavable targets, the variants separate the DNA strands in the PAM-proximal region upon binding to the target, however, the mutations inhibit the effective full-length separation of the two strands of the target DNA, and thus they also prevent the formation of the cleavage-competent conformation of SpCas9 52,55,57 . HeF-SpCas9, which has the highest delity and lowest average cleavage activity among these variants, binds to the targets of WT-SpCas9 without being able to cleave most of them: in contrast to the wild type nuclease 53,54,55 , it fails to effectively separate the strands of the target DNA at full length and to acquire the cleavage-competent conformation 43,45,52 . Based on these considerations, we expected that HeF-ABE, which lacks a nicking activity, would show a rather dABE-like activity on many targets and a dABE-like activity pro le.…”

“…In fact, dSpCas9 in dABE separates the strands of the target DNA at full length 57 , facilitating the action of the deaminase, which is active only on single-strand DNA 8 . However, when increased delity nucleases are simply bound to the DNA without being able to cleave or nick the target, they separate the two strands of the target DNA at its seed region only, while the PAM distal region is separated less e ciently, rather transiently 52,55,57 . We speculate that in such cases, the deaminase is able to proceed in dABE, but not in HeF-ABE.…”

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

“…Unpaired nucleotides at the 5′ end of the spacer sequence can influence Sp Cas9 activity in a protein and target site-dependent manner ( 16 , 17 ). For WT Cas9, one or two additional 5′ guanines lead to increased specificity due to reduced unwinding promiscuity.…”