The CRISPR toolbox is continuously evolving, notably in the expansion of Cas enzymes available for gene editing. Both the discovery of new Cas enzymes and the enhancement of previously known Cas enzymes are adding to the repertoire of available nucleases. This week, a group of researchers from Massachusetts General Hospital (MGH) has published an enhanced variant of the CRISPR-Cas12a enzyme that has increased activities and improved targeting ranges for gene and epigenetic editing.

The paper, “Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing” was published in Nature Biotechnology.

Cas12a (formerly Cpf1) nucleases have shown promise in multiple gene editing applications, however, carry the limitation of a requirement for a longer protospacer adjacent motif (PAM)—specifically of the form 5’-TTTV (where V is an A, C, or G.) This particular qualification restricts the targeting roughly sixfold relative to the commonly used Streptococcus pyogenesCas9 (SpCas9.)

The group from the lab of Keith Joung, MD, PhD, used structure-guided protein engineering to create variants of the Acidaminococcus sp. Cas12a with an expanded PAM recognition profile. The team created “ten variants bearing single amino acid substitutions to positively charged arginine residues that might be expected to alter or form novel PAM proximal DNA contacts.” Based on their findings, the team choose one triple mutant with the most expanded PAM targeting range, referred to as enhanced AsCas12a (enAsCas12a.) When compared with wild type, this variant showed a substantially expanded targeting range, enabling targeting of many previously inaccessible PAMs and an average two-fold higher genome editing activity on sites with canonical TTTV PAMs.

“These combined enhancements make enAsCas12a the most active and targetable Cas12a enzyme available to date,” noted first author Ben Kleinstiver, PhD, a former post-doc in the Joung lab who currently has his own group in the Center for Genomic Medicine at MGH.

Kleinstiver told GEN that, “enAsCas12a addresses several of the limitations of Cas12a by improving targeting range ~7–8-fold compared to wild-type Cas12a and by enhancing on-target activity by ~2–3-fold; these improvements collectively make the Cas12a platform more comparable to SpCas9 across these properties.”

“We also showed that the improvements associated with enAsCas12a translate into primary human T cells, where we demonstrated far more potent editing (2–3-fold improved again compared to wild-type AsCas12a) and the ability of enAsCas12a to potently target sites previously inaccessible to Cas12a enzymes,” noted Kleinstiver.

This report comes on the heels of two other publications reporting new Cas enzymes. In late January, a report from the lab of CRISPR co-discoverer Feng Zhang, PhD, professor of neuroscience at MIT, reported a newly engineered Cas12b enzyme with increased functionalities. The enhancements were an important advance as Cas12b’s temperature restrictions were a significant barrier to the utility of the enzyme in physiologically relevant conditions. Similarly, the lab of Jennifer Doudna, PhD, University of California, Berkeley recently revealed the underlying mechanisms of a previously undescribed, fundamentally distinct genome-editing platform named CasX. All three enzyme families (Cas12a, Cas12b, and CasX (Cas12e) have slightly different properties. To this end, Kleinstiver noted that it is “likely that each has their own advantages.”

Not only might one Cas be more applicable than another for a particular situation, but, more tools in the toolbox are particularly useful to researchers performing multiple genome editing tasks at one time. For those who, for example, want to edit DNA concurrently with epigenome and gene regulation modifications, the more the merrier.

 

 

 

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