A recent study reveals why the Cas12a genome-editing enzyme is more specific than the widely used Cas9. For Cas12a to cut, the guide RNA it carries must more closely match the sequence of the DNA target than with Cas9. The enzyme's specificity may make it a better choice for gene-editing applications.

A recent study uncovers why the lesser-known Cas12a nuclease is more specific than the more commonly used Cas9 enzyme for CRISPR-based genome editing (Mol Cell 2018;71:816–24). The results may lead to wider application of Cas12a for research and therapies.

Cas9 was the first CRISPR editing enzyme discovered. Most researchers still rely on it for lab studies, as do all current clinical trials using the editing technology. However, Cas9 can make off-target alterations, and a recent study revealed that it can also cause large deletions and additional genomic damage around the cutting site (Nat Biotechnol 2018;36:765–71). Other studies suggest that Cas12a, a related but less-studied enzyme, is more precise than Cas9.

To find out why, a team from The University of Texas at Austin led by Rick Russell, PhD, and Ilya Finkelstein, PhD, measured the rate of DNA editing by Cas12a in vitro. During CRISPR editing, a section of the guide RNA molecule carried by the enzyme recognizes and binds to a complementary DNA sequence, forming a hybrid structure called the R-loop. The nuclease then cuts both DNA strands.

One hypothesis is that Cas12a can be more selective because of the way the guide RNA binds to the target DNA. The researchers tested this idea by determining the effect of DNA–RNA sequence mismatches at different positions. Guide RNAs seek stretches of DNA that are around 20 bases long. For Cas9, the first 10 or so nucleotides of the DNA target need to match the corresponding nucleotides of the guide RNA, but matches at the remaining positions are less important for binding.

graphic

This illustration shows the Cas12a nuclease bound to DNA (red and white).

However, Russell, Finkelstein, and colleagues found that mismatches at the distal end of the DNA sequence are more important for Cas12a than they are for Cas9. They could cause the R-loop to dissociate even after it had mostly formed. The team's results suggest that Cas12a's increased discrimination “comes from having substantial specificity for almost the entire length of the guide RNA,” says Russell.

“They did a really good job of untangling why Cas12a has been observed to exhibit higher specificity than run-of-the-mill Cas9,” says Mitchell O'Connell, PhD, of the University of Rochester Medical Center in New York, who wasn't connected to the study. John van der Oost, PhD, of Wageningen University in the Netherlands, agrees. “The specificity of Cas12a is significantly better than Cas9, and they used a very elegant method to demonstrate that.”

The results may prompt more researchers to adopt Cas12a for research and for new treatments, scientists say. “Cas12a should be considered a front-runner for gene-editing applications,” Russell says. The study's results may enable researchers to engineer better versions of the enzyme, adds Dipali Sashital, PhD, of Iowa State University in Ames. “By understanding the mechanism for its higher specificity, we might be able to tweak it further.” –Mitch Leslie

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