Enhanced Acidaminococcus sp. Cas12a (enAsCas12a)

Characteristics

Carries the E174R, S542R, and K548R substitutions in wild-type AsCas12a, relaxing PAM recognition from the canonical TTTV to also include TTYN, VTTV, and TRTV, an approximately sevenfold expansion in targetable genomic sites. Cleaves both DNA strands with a single RuvC-like domain, producing the same staggered double-strand break with a 5' overhang as wild-type AsCas12a. On-target editing activity at canonical TTTV sites is about twofold higher than wild-type AsCas12a. This gain comes with slightly increased off-target activity relative to wild-type AsCas12a; the derivative enAsCas12a-HF1 adds N282A to bring specificity back toward wild-type levels.

Applications: Multiplex gene editing, dCas12a-based endogenous gene activation, and C-to-T base editing in HEK293T cells; functions as a ribonucleoprotein complex in primary human T cells. Broadened PAM access supports CRISPR-directed evolution screens for drug-resistance mutations at genomic sites inaccessible to SpCas9 or wild-type AsCas12a.

Limitations: Retains AsCas12a's single RuvC-like active site, so mutating it eliminates cleavage of both strands rather than yielding a nickase. Shows slightly higher off-target activity than wild-type AsCas12a at some non-canonical PAM sites; the enAsCas12a-HF1 derivative (E174R/N282A/S542R/K548R) reduces off-target cleavage toward wild-type levels by GUIDE-seq.

References

1. Kleinstiver et al. (2019). Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat Biotechnol - Kleinstiver 2019 enCas12a
2. Neggers et al. (2020). enAsCas12a enables CRISPR-directed evolution to screen for functional drug resistance mutations in sequences inaccessible to SpCas9. Mol Ther - Neggers 2020 enAsCas12a Screening