Cas9, a widely used CRISPR-based gene-editing tool, has been altered to be thousands of times less likely to target the erroneous stretch of DNA while staying just as effective as the previous version, perhaps making it considerably safer.
One of the major drawbacks of using CRISPR-based gene editing on humans is that the molecular machinery can sometimes make changes to the wrong part of a host’s genome, posing the risk that repairing a genetic mutation in one part of the genome could result in the creation of a dangerous new mutation in another.
However, researchers at The University of Texas at Austin have modified a critical component of the widely used CRISPR-based gene-editing tool Cas9 to be thousands of times less likely to target the erroneous stretch of DNA while being just as effective, perhaps making it considerably safer. The research is detailed in a report published in the journal Nature today.
Kenneth Johnson, a professor of molecular biosciences and co-senior author of the work with David Taylor, an associate professor of molecular biosciences, stated, “This truly might be a game changer in terms of a broader use of the CRISPR Cas systems in gene editing.” Postdoctoral fellows Jack Bravo and Mu-Sen Liu are co-first authors on the work.
Other laboratories have altered Cas9 to limit off-target interactions, but all of these variants have improved accuracy at the expense of speed thus far. This new variant, termed SuperFi-Cas9, is 4,000 times less likely to cut off-target sites while still being as rapid as naturally occurring Cas9. According to Bravo, the many lab-generated variants of Cas9 may be compared to different types of self-driving automobiles. Although the majority of models are quite safe, they do have a peak speed of 10 miles per hour.
“They’re safer than naturally occurring Cas9, but at a significant cost: they’re exceedingly sluggish,” Bravo said. “SuperFi-Cas9 is similar to a self-driving automobile that has been designed to be exceedingly safe while yet being able to go at full speed.”
The researchers have shown that SuperFi-Cas9 can be used on DNA in test tubes thus far. They’re currently working with other scientists to see whether SuperFi-Cas9 can be used to modify genes in live cells. They’re also focusing on making Cas9 that’s even safer and more active.
CRISPR-based gene editing technologies are inspired by naturally existing bacterial systems. A Cas9 protein floats about in nature, looking for DNA with a highly precise 20-letter pattern, similar to the X on a pirate map that means “dig here.” Cas9 will sometimes go ahead and dig in even if the majority of the letters are accurate, except for those in positions 18 through 20. This is referred to as a mismatch, and it may lead to devastating results in gene editing.
Taylor and Johnson utilized a cryo-electron microscope at the Sauer Structural Biology Lab to acquire images of Cas9 in motion as it interacted with this mismatched DNA in a method termed kinetics-guided structure determination.
Cas9, they discovered, has a finger-like structure that swoops in and grabs on to the DNA, making it operate as if it were the proper sequence, rather than giving up and going on when it detects this sort of mismatch in positions 18 through 20. A mismatch normally causes the DNA to become floppy, but this finger-like structure stabilizes it.
“It’s like if one of the legs of a chair came off and you simply duct glued it back together,” Bravo said. “It could still be used as a chair, although it would be a little shaky. It’s a shady arrangement.”
Cas9 doesn’t perform the subsequent steps required to cut the DNA and make modifications if it doesn’t have the extra stability in the DNA. No one had ever seen this additional finger doing this function previously.
“This was something I could never have imagined in a million years happening in my head,” Taylor added.
They altered the additional finger on Cas9 based on this knowledge, such that instead of stabilizing the region of the DNA containing the mismatch, the finger is pushed away from the DNA, preventing Cas9 from completing the cutting and editing process. SuperFi-Cas9 is the outcome, a protein that cuts the proper target exactly as well as naturally occurring Cas9, but is considerably less likely to cut the erroneous target.
Grace Hibshman, Tyler Dangerfield, Kyungseok Jung, and Ryan McCool, all at The University of Texas at Austin, are also writers.
Bravo, Liu, Hibshman, Dangerfield, Johnson, and Taylor are co-inventors on a patent application based on this research that covers innovative Cas9 designs. The UT Austin Office of Technology Commercialization is in charge of the intellectual property and is looking for industry partners to assist realize the technology’s enormous potential.
The Welch Foundation and the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation both contributed to this project. The Cancer Prevention and Research Institute of Texas supports Taylor as a CPRIT scholar. Taylor is also funded by the David Taylor Excellence Fund in Structural Biology, which was established thanks to Judy and Henry Sauer’s generosity.