New SuperFi-Cas9 Increases Fidelity Without Sacrificing Speed
Updated: Mar 16
CRISPR–Cas9 has limitations. One of the most concerning, in regards to using genome editing for therapeutic applications for humans, is off-target DNA cleavage. Although some Cas9 variants have been developed that have improved mismatch discrimination, they suffer from reduced rates of on-target DNA cleavage.
Because the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood, a group of scientists sought to determine the structure of Cas9 at different stages of mismatch cleavage using kinetics-guided cryo-electron microscopy. In doing so, they provided a molecular blueprint for the design of next-generation high-fidelity Cas9 variants that reduce off-target DNA cleavage while retaining efficient cleavage of on-target DNA. Using that blueprint, they designed a high-fidelity variant that retains wild-type on-target cleavage rates which they call “SuperFi-Cas9.”
The work is described in a paper published in Nature titled, “Structural basis for mismatch surveillance by CRISPR–Cas9.”
“This really could be a game-changer in terms of a wider application of the CRISPR Cas systems in gene editing,” said Kenneth Johnson, a professor of molecular biosciences at the University of Texas (UT), Austin.
SuperFi-Cas9 is 4,000 times less likely to cut off-target sites but just as fast as naturally occurring Cas9. Jack Bravo, PhD, a postdoctoral fellow in the lab of David Taylor, PhD, assistant professor in the department of molecular biosciences at UT Austin, said you can think of the different lab-generated versions of Cas9 as different models of self-driving cars. Most models are really safe, but they have a top speed of 10 miles per hour. “They’re safer than the naturally occurring Cas9, but it comes at a big cost: They’re going extremely slowly,” said Bravo. “SuperFi-Cas9 is like a self-driving car that has been engineered to be extremely safe, but it can still go at full speed.”