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CRISPR Researchers Engineer More Efficient Cas12a Variants for Genetic, Epigenetic, Base Editing

NEW YORK (GenomeWeb) – In a study published today in Nature Biotechnology, researchers in Keith Joung's lab at Massachusetts General Hospital described their efforts to engineer enhanced variants of the Cas12a nuclease that can target a wider range of protospacer adjacent motifs (PAM), have enhanced editing activity, and have reduced off-target effects.

The group's Acidaminococcus sp. Cas12a variant (enAsCas12a) has a substantially expanded targeting range, allowing it to target many previously inaccessible PAMs as it does not require an extended TTTV PAM like AsCas12a does. Further, the researchers found that enAsCas12a exhibited an average two-fold higher genome editing activity on sites with canonical TTTV PAMs compared to wild-type AsCas12a, and they successfully grafted a subset of mutations from enAsCas12a onto other previously described AsCas12a variants to enhance their activities.

"EnAsCas12a improves the efficiency of multiplex gene editing, endogenous gene activation and C-to-T base editing, and we engineered a high-fidelity version of enAsCas12a (enAsCas12a-HF1) to reduce off-target effects," the authors wrote. "Both enAsCas12a and enAsCas12a-HF1 function in HEK293T and primary human T cells when delivered as ribonucleoprotein (RNP) complexes. Collectively, enAsCas12a provides an optimized version of Cas12a that should enable wider application of Cas12a enzymes for gene and epigenetic editing."

In general, Cas12a nucleases — including AsCas12a and Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a) — recognize target sites with T-rich PAMs, require only a single short CRISPR RNA (crRNA) of about 40 nucleotides to program target specificity, and possess RNase activity that enables multiplex targeting through poly-crRNA transcript processing. Although Cas12a enzymes have shown utility for multiplex gene editing, gene activation, and combinatorial library screens, one constraint compared to Cas9 is their requirement for the longer5'-TTTV PAM.

For their study, the researchers used structure-guided protein engineering to expand the PAM recognition of Cas12a nucleases, and engineered 10 variants that bore single amino-acid substitutions to positively charged arginine residues that they believed could alter or form novel PAM proximal DNA contacts. Four of the 10 variants displayed higher gene editing activities in human cell lines on sites with canonical and non-canonical PAMs relative to wild-type AsCas12a. Further testing showed that two variants exhibited the highest editing activities on sites with non-canonical PAMs while still retaining robust activities on a canonical PAM site.

The researchers' analyses showed that the enAsCas12a variant expanded the targeting range by approximately sevenfold. They also compared the activities of AsCas12a, enAsCas12a and LbCas12a programmed with poly-crRNA arrays targeted to three endogenous genes in human cells and observed comparable or higher editing with enAsCas12a relative to AsCas12a and LbCas12a.

When the team looked at the off-target effects of the engineered variant, they found that it had more off-targets than the wild-type AsCas12a. In order to improve on the specificity of enAsCas12a, they then engineered a high-fidelity variant containing substitutions in amino acid residues expected to make non-specific contacts to DNA. Among the variants they examined, the researchers found that enAsCas12a-N282A exhibited the greatest improvement in single mismatch intolerance while retaining on-target activity similar to enAsCas12a.

To more thoroughly determine if the N282A substitution affected on-target activity, the team compared enAsCas12a and enAsCas12a-HF1 by performing in vitro cleavage assays to assess temperature tolerance, which revealed similar cleavage profiles among enAsCas12a, enAsCas12a-HF1, and LbCas12a. The researchers also compared the on-target activities of enAsCas12a and enAsCas12a-HF1 and found that they had similar on-target activities across six sites with TTTV PAMs and on 17 target sites with various non-canonical PAMs.

"The enhanced AsCas12a variants described herein substantially improve the targeting range, on-target activities, and fidelity of Cas12a nucleases, which are properties that are important for multiplex gene editing, epigenetic editing, cytosine base editing, and gene knockout in primary human T cells," the authors concluded.

"Our results provide an important proof-of-concept that the on-target potency of CRISPR enzymes can be augmented through engineering, a strategy that may be extensible to other CRISPR nucleases," they added. "Future structural studies will be helpful to characterize the roles of the substitutions in our AsCas12a variants, and additional work may be required to determine whether the potency of enAsCas12a and enAsCas12a-HF1 RNPs are sufficient for therapeutic applications."

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