When good isn’t good enough

University of Texas researchers think they have a way to make the gene-editing tool CRISPR work better

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AUSTIN, Texas—The gene-editing technology known as CRISPR (which stands for clustered regularly interspaced short palindromic repeats) continues to make gains both in the editing of non-human genomes for experimental purposes and also coming closer to being a potential therapeutic modality for humans. But as useful as CRISPR has been for quickly and affordably modifying the genetics of living creatures, many keep looking for ways to increase its utility and ensure its safety. And to that end, scientists at the University of Texas at Austin believe that they have identified a relatively easy upgrade for CRISPR—which nature intended as a mechanism for bacteria to defend against viruses—that would mean more accurate gene editing and also the increased safety that could make the technology appropriate for use in humans.
And they did so by moving away from Cas9, which has dominated most of the lab research and headlines, and looking at the Cas12a protein instead.
In CRISPR/Cas9, the system consists of two key molecules that introduce a change into the targeted DNA. The first is the enzyme Cas9, which acts like a pair of “molecular scissors” to snip the twin strands of DNA that represent the subject of interest at a specific location, thereby allowing bits of DNA to be added or deleted. The second is the guide RNA, a small piece of pre-designed RNA sequence that guides Cas9 to the right part of the genome.
In the work carried out by the University of Texas researchers, they came to the conclusion that Cas9, while it was the first enzyme for CRISPR to be discovered and the most popular so far, has less effectiveness and precision than Cas12a. Their findings were published Aug. 2 in the journal Molecular Cell under the title “Kinetic Basis for DNA Target Specificity of CRISPR-Cas12a.”
Precision is important, because the risk of editing the wrong part of the genome is one of the stumbling blocks for CRISPR as a human therapeutic, and also can disrupt plant and animal health in unintended ways during experiments or lead to incorrect changes that would skew or ruin an experiment.
“The overall goal is to find the best enzyme that nature gave us and then make it better still, rather than taking the first one that was discovered through historical accident,” said Ilya Finkelstein, an assistant professor of molecular biosciences and a co-author of the study.
As the researchers note, CRISPR systems found in nature sometimes target the wrong spot in a genome, which in humans could be disastrous—for example, failing to correct for a genetic disease and instead turning healthy cells into cancerous cells. They add that while some previous studies have hinted that Cas12a is “choosier” than Cas9, that work was inconclusive; their work, they say, shows that Cas12a is indeed a more precise gene-editing tool than Cas9.
The team, led by graduate student Isabel Strohkendl and Prof. Rick Russell, reportedly found that Cas12a is more accurate because, as the university explains, it binds like Velcro to a genomic target, whereas Cas9 binds to its target more like super glue.
With Cas9, each base pair sticks together tightly, but the problem is that Cas9 pays attention to the first seven or eight base-pair “letters” in the genomic target, and pays less attention as the process goes on. As such, it might overlook a mismatch later in the process that would lead it to edit the wrong part of the genome.
For Cas12a, however, the bonds along the way as it attempts to edit the genome are relatively weak, and it takes a good match all along the strip for the two sides to hold together long enough to make an edit. That makes it much more likely that it will edit only the intended part of the genome.
“It makes the process of base-pair formation more reversible,” Russell explained. “In other words, Cas12a does a better job of checking each base pair before moving on to the next one. After seven or eight letters, Cas9 stops checking, whereas Cas12a keeps on checking out to about 18 letters.”
The researchers acknowledge that Cas12a still isn’t perfect, but they went further in their work to suggest methods by which Cas12a could be improved further.
“On the whole, Cas12a is better, but there were some areas where Cas12a was still surprisingly blind to some mispairing between its RNA and the genomic target,” Finkelstein said. “So what our work does is show a clear path forward for improving Cas12a further.”
The researchers are currently using their insights in a follow-on project designed to engineer an improved Cas12a.

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