Scientists at The University of Texas at Austin have redesigned a key component of a widely used CRISPR-based gene-editing tool, called Cas9, to be thousands of times less likely to target the wrong stretch of DNA while remaining just as efficient as the original version, making it potentially much safer.
Other labs have redesigned Cas9 to reduce off-target interactions, but so far, all these versions improve accuracy by sacrificing speed. SuperFi-Cas9, as this new version has been dubbed, is 4,000 times less likely to cut off-target sites but just as fast as naturally occurring Cas9. Bravo says 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.”
So far, the researchers have demonstrated the use of SuperFi-Cas9 on DNA in test tubes. They’re now collaborating with other researchers who plan to test SuperFi-Cas9 for gene editing in living cells. They’re also working to develop still safer and more active versions of Cas9.
Taylor and Johnson developed a technique called kinetics-guided structure determination that used a cryo-electron microscope in the Sauer Structural Biology Lab to take snapshots of Cas9 in action as it interacted with this mismatched DNA.
They were surprised to discover that when Cas9 encounters this type of mismatch in positions 18 through 20, instead of giving up and moving on, it has a finger-like structure that swoops in and holds on to the DNA, making it act as if it were the correct sequence. Normally, a mismatch leaves the DNA a bit floppy; this finger-like structure stabilizes it.
“It’s like if you had a chair and one of the legs was snapped off and you just duct taped it together again,” Bravo said. “It could still function as a chair, but it might be a bit wobbly. It’s a pretty dirty fix.”
Based on this insight, they redesigned the extra finger on Cas9 so that instead of stabilizing the part of the DNA containing the mismatch, the finger is instead pushed away from the DNA, which prevents Cas9 from continuing the process of cutting and editing the DNA. The result is SuperFi-Cas9, a protein that cuts the right target just as readily as naturally occurring Cas9, but is much less likely to cut the wrong target.
CRISPR–Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood. Although Cas9 variants with greater discrimination against mismatches have been designed these suffer from substantially reduced rates of on-target DNA cleavage. Here we used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. We observed a distinct, linear conformation of the guide RNA–DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA–DNA duplex conformation facilitates DNA cleavage, we observe that substrates that contain mismatches distal to the protospacer adjacent motif are stabilized by reorganization of a loop in the RuvC domain. Mutagenesis of mismatch-stabilizing residues reduces off-target DNA cleavage but maintains rapid on-target DNA cleavage. By targeting regions that are exclusively involved in mismatch tolerance, we provide a proof of concept for the design of next-generation high-fidelity Cas9 variants.
SOURCES- University of Texas, Nature
Written by Brian Wang, Nextbifuture.com
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