The roughly half a dozen Cas9 enzymes available for DNA editing scratch the surface of the panoply of Cas enzymes that exist in bacteria. Today, a team led by CRISPR co-discoverer Feng Zhang, PhD, professor of neuroscience at MIT, together with colleagues at the Broad Institute, Harvard University, and the National Institutes of Health, demonstrated that a relatively new Cas enzyme, Cas12b, can be engineered to target and precisely nick or edit the genomes of human cells.

“This work puts on the scene another powerful genome engineering tool that will complement and, in some respects, might surpass Cas9 and Cas12a,” notes Eugene Koonin, PhD, senior investigator in the Evolutionary Genomics Research Group of the National Library of Medicine/National Center for Biotechnology Information at the NIH, and co-author on the paper.

The paper, entitled “Engineering of CRISPR-Cas12b for human genome editing,” was published today in Nature Communications.

Rodolphe Barrangou, PhD, professor at North Carolina State University, tells GEN that “this is not the discovery of Cas12b, per se.” Indeed, the family of Cas12b enzymes was first described several years ago and has been reported on since. In 2017, the lab of another CRISPR co-discoverer, Jennifer Doudna, PhD, professor at the University of California, Berkeley, used the Cas12b from Alicyclobacillus acidoterrestris and a second Cas12b from Alicyclobacillus acidiphilus and was described last fall by Wei Li’s team at the Chinese Academy of Sciences in Beijing.

But, the previously described Cas12b enzymes had major hurdles to being useful—namely a requirement for functioning at a high temperature. In describing the Cas12b from Bacillus hisashii (BhCas12b), Jonathan Strecker, PhD, a postdoctoral fellow in the Zhang Lab, and colleagues have made the enzyme more “valuable and useful” by “developing and enhancing a specific Cas12b with increased potential and functionalities to perform genome editing in human cells in physiologically relevant conditions,” notes Barrangou.

Strecker and colleagues screened a variety of previously uncharacterized orthologs for high activity at human body temperature, choosing the BhCas12b ortholog, and then performed structure-guided protein engineering to further improve its DNA cutting activity, notes Patrick Hsu, PhD, a principal investigator at the Salk Institute for Biological Studies (and former graduate student in the Zhang lab.)

“We searched for inspirations from nature,” Zhang said in a press release from MIT. “We wanted to create a version of Cas12b that could operate at lower temperatures, so we scanned thousands of bacterial genetic sequences, looking in bacteria that could thrive in the lower temperatures of mammalian environments.”

Hsu tells GEN that “the coolest thing about this paper is the rational engineering to significantly improve the nuclease activity,” adding that “it would be tremendously exciting to see if similar efforts could be used to improve SpCas9 efficiency, although this has not yet been published, perhaps because SpCas9 already works very well at 37°C.” Another notable finding, according to Hsu, was that their optimized tool can efficiently target endogenous genes in primary human T cells ex vivo, noting that a head-to-head comparison with S. pyogenes Cas9 would have been great to see. Also, that BhCas12b is more specific out of the box than SpCas9, although many high specificity variants of SpCas9 have been created over the last few years. Similar approaches are likely to further improve Cas12b specificity which could lead to “much lower cleavage at off-target sites,” adds Dipali Sashital, PhD, assistant professor at Iowa State University.

“It was surprising and impressive that the engineered Cas12b has comparable genome editing activity to Cas9 and Cas12a,” Sashital tells GEN. She adds that the size difference between Cas9 and Cas12b is also important to note. “BhCas12b is much smaller than Streptococcus pyogenes Cas9, so it can be delivered by adeno-associated virus (AAV),” adding that, although smaller orthologs of Cas9 are available (such as from Staphylococcus aureus) that can also be delivered by AAV, it is useful to have more small Cas endonucleases to increase the number of protospacer adjacent motif (PAM) sequences that can be targeted.

Another notable distinction, according to Barrangou, is that the Cas12b PAM is AT rich, which increases the precision with which you can hit a target of interest which increases the overall landscape of sequence targeting and sequence precision. In addition, the mutation outcome landscape—whereby they frequently obtain a 5–15 nucleotide range of deletions—would provide an advantage when making gene knockouts.

The team engineered the protein to favor the creation of double-strand breaks, which are required for genome editing. This, to Sashital’s knowledge, is the first study to improve the rate of double-strand break formation, as opposed to nicking, which compellingly shows how understanding the mechanism of Cas endonuclease function allows us to continue to improve these tools. In addition to developing a new genome editing tool, this work shows that with enough exploration and engineering of different Cas endonucleases, we should be able to continue to expand the number of CRISPR tools that are available to the research community.

The idea of “more is better” is seconded by Barrangou, noting that this addition to the CRISPR toolbox enables users to more readily perform multiplex applications, especially for people who want to focus on gene circuits and cell engineering—to redirect the ability of an organism to have different biochemical pathway outcomes. In order to alter both the genome and the transcriptome, you need more than one tool. Not only that, Barrangou says, “if you want to do these things concurrently, you need two tools that are fully orthogonal, that don’t cross talk.” Barrangou can see a world where the genome is edited with Cas9 and then Cas12b is used to drive upregulation. Even more, another flavor of Cas12—Cas12a—could be used at the same time to drive downregulation.” He adds that the epigenome could also be changed, with the addition of yet another tool. So, access to more tools that are fully orthogonal will allow for more functional multiplexing. Although these goals may be achievable now, using the cadre of Cas9 enzymes currently available, there is sometimes cross talk between them which would be minimized by new families of enzymes.

The experts agree that this is not, by any stretch, the end of the Cas-discovery road. “We have barely tapped the functional diversity of CRISPR systems existing in prokaryotes,” notes Koonin. And Barrangou agrees that “a very small proportion of the overall CRISPR-Cas landscape has been fully characterized to date.” Perhaps the most exciting aspect of this work is the promise of the continued expansion of the CRISPR toolkit.