High-fidelity Cas12Max, an engineered Cas12i-family nuclease (hfCas12Max)

Characteristics

Single-RuvC-domain type V-I nuclease that autonomously processes its own pre-crRNA and preferentially nicks the non-target DNA strand before cutting the target strand, characteristic of the Cas12i family. Carries N243R (PAM-interacting domain), E336R (REC domain), and D892R (RuvC domain) substitutions over parental xCas12i. Recognizes broad 5'-TN and 5'-TNN PAMs, including TTN, ATN, GTN, and CTN, and reaches an average 70% indel efficiency across TTR reporter loci in HEK293T cells, exceeding SpCas9, LbCas12a, and Ultra-AsCas12a tested under matched conditions. Shows reduced indel frequency at six identified off-target sites relative to the single-mutant Cas12Max while retaining comparable on-target activity.

Applications: Ex vivo TRAC knockout in human CD3+ T cells via ribonucleoprotein delivery, reaching approximately 90% editing with approximately 80% viability. In vivo liver editing via lipid-nanoparticle-delivered mRNA, reaching approximately 70% indel frequency at the murine Ttr locus at 0.3-0.5 mg/kg. Nuclease-dead fusions support adenine and cytosine base editing (dCas12Max-ABE, dCas12Max-CBE) in HEK293T cells.

Limitations: Requires a 5'-TTN-type PAM, though broader than most natural Cas12i orthologs. Reported in a single 2023 study; independent replication and long-term in vivo safety data are not yet available. Off-target reduction was benchmarked against a small panel of predicted sites rather than genome-wide profiling.

References

1. Zhang et al. (2023). An engineered xCas12i with high activity, high specificity, and broad PAM range. Protein Cell - Zhang 2023 hfCas12MAX