In the world of gene-editing, CRISPR and Cas9 usually go hand in hand, but that might not necessarily be the best pairing. The Cas9 protein has proven effective over the last few years, but alternatives, such as Cas12a and Cas12b, are quickly emerging. Now, researchers at UC Berkeley have tested a new candidate, CasX, which seems to have a few advantages of its own.

The CRISPR gene-editing system was inspired by the tools bacteria use to defend themselves. When the bugs encounter a new virus that poses a threat to them, they'll use an enzyme like a pair of genetic scissors, snipping out a small segment of the attacker's DNA and storing it, so the bacteria remembers how to fight it next time.

A few years ago scientists adapted that technique to develop the CRISPR tool, which uses certain enzymes to cut out specific DNA sequences and replace them with something more beneficial. This powerful system is opening brand new ways to treat disease in humans, breed better animals, make crops more nutritious or manipulate microorganisms.

Since the beginning, Cas9 has been the enzyme doing all the hard work, but more recently Cas12 variants are emerging as efficient alternatives. And now, CasX has joined them. The UC Berkeley team discovered CasX two years ago in bacteria commonly found in groundwater, and while it seemed promising it had never been tested outside of its host organism.

In new tests, the researchers have found that it is effective at editing the human genome, as well as that of E. coli. CasX works in much the same way as Cas9 – it can be made to target specific sequences, cut through double-stranded DNA, and bind to DNA in order to regulate genes.

But CasX has a few advantages over its predecessors. The protein is much smaller, which helps it get inside cells easier, and since it was isolated from a bacteria species that isn't found in humans, it's less likely to trigger an immune response in patients, which has been a long-time concern of CRISPR-Cas9.

"The immunogenicity (likelihood of triggering an immune response), delivery and specificity of a genome-editing tool are all vitally important," says Benjamin Oakes, co-lead author of the study. "We're excited about CasX on all of these fronts."

Interestingly, although both enzymes perform the same function, the team says they're different enough in molecular makeup and shape that they seem to have evolved completely independently of each other. Studying these proteins could help inform future designs.

"The first thing that jumps out is how the highly unique domains accomplish similar roles to what we have seen with other RNA-guided DNA-binding proteins," says Oakes. "CasX's minimal size, with no fat on the bone, helps to clearly demonstrate there is a basic recipe that nature uses. Understanding this recipe will help us to better evolve and engineer genome editing tools for our purposes rather than nature's."

The research was published in the journal Nature.

Source: UC Berkeley