NEW YORK — Researchers have developed a new version of the Cas9 enzyme used in genome editing that is less likely to make off-target cuts but that still works as quickly as the wild-type enzyme.
A stumbling block to using CRISPR-Cas9 genome editing in the clinic is the possibility of off-target cleavage by Cas9 that could induce new mutations rather than repair existing ones. Other forms of Cas9 have been developed with the aim of improving its accuracy, but researchers from the University of Texas at Austin said those tweaks have largely come at the expense of efficiency.
By using kinetics-guided cryo-electron microscopy, the Texas team examined the shape of Cas9 at different points mid-mismatch cleavage. Through this, they uncovered a structural feature that stabilizes the Cas9-mismatched DNA complex that enables the enzyme to cleave. As they reported in Nature on Wednesday, redesigning that stabilizing feature leads to an enzyme, dubbed SuperFi-Cas9, that cleaves the correct target as often as wild-type Cas9 but is less likely to cut an incorrect one.
"This really could be a game changer in terms of a wider application of the CRISPR Cas systems in gene editing," co-senior author Kenneth Johnson, a professor of molecular biosciences at UT-Austin, said in a statement.
He and his colleagues used a cryo-EM-based approach to generate pictures of what the Cas9-mismatch DNA cleavage structure looks during the cleavage process.
They focused at first on samples of Cas9 interacting with mismatches at 12 to 14 base pairs distal from the protospacer-adjacent motif. In such samples, Cas9 activation occurs but cleavage is slower than usual.
Specifically, the researchers identified two distinct Cas9 conformations in these samples. A linear duplex conformation largely arose in the initial sample with short cleavage times, while that conformation plus the canonical kinked duplex conformation was present in the samples with longer cleavage times. Based on this, the researchers suspected that the linear duplex conformation represents an early intermediate of Cas9 before the target-strand-cleaving HNH endonuclease domain comes into place.
They further examined Cas9 samples interacting with mismatches at 18 to 20 base pairs distal from the PAM, mismatches that are generally more tolerated. Using cryo-EM, the researchers again found both linear and kinked conformations arise but also noted the presence of two linker domains in the kinked conformation that connect the HNH to the rest of the Cas9 and lock it into conformation. In particular, a reorganization of a loop in the RuvC domain appears to stabilize the interaction and could enable Cas9 activation despite the mismatch.
"It's like if you had a chair and one of the legs was snapped off and you just duct-taped it together again," co-first author Jack Bravo, a postdoc at UT-Austin, said in a statement. "It could still function as a chair, but it might be a bit wobbly. It's a pretty dirty fix."
The finding also suggested to the researchers a way to engineer a new Cas9 enzyme. In their SuperFi-Cas9, all of the seven stabilizing residues are mutated to aspartic acid. SuperFi-Cas9, they reported, cuts on-target DNA at a rate similar to that of wild-type Cas9, while cleavage of DNA mismatches at 18 to 20 base pairs distance was 500-fold slower.
The researchers noted that they have so far only tested SuperFi-Cas9 on DNA in test tubes but are now working with their collaborators to test it in living cells.