Engineered miniature CRISPR-Cas12f nuclease (CasMINI)

Characteristics

529-amino-acid nuclease engineered from Un1Cas12f1, an archaeal Cas12f (Cas14) protein that shows no detectable activity in mammalian cells in its unmodified form. A cluster of protein substitutions (D143R/T147R/E151A) together with a redesigned single-guide RNA scaffold restore robust activity in human cells. Recognizes a 5'-TTTR PAM; TTTA and TTTG support activity while TTTC does not. In HEK293 cells, dCasMINI-VPR activates endogenous genes (IFNγ, CD2, CXCR4, HBB) at levels comparable to dCas12a-VPR, with no off-target activation detected by whole-transcriptome analysis. Nuclease activity produces predominantly large deletions 20-30 bp distal to the PAM, distinct from SpCas9-style small indels.

Applications: Gene activation of endogenous human loci (IFNγ, CD2, CXCR4, HBB) via dCasMINI-VPR, adenine base editing via dCasMINI-ABE, and gene disruption via nuclease-active CasMINI, all demonstrated in HEK293 human cells. Its small size (529 aa) suits AAV-packaging-constrained delivery contexts where SpCas9 or Cas12a leave little room for a promoter and polyA signal.

Limitations: Requires a 5'-TTTR PAM (TTTC is inactive), more restrictive than SpCas9's NGG. Nuclease activity produces large deletions (20-30 bp) rather than small indels, unsuitable when a precise small edit is required. Base editing shows bystander adenine conversions near the target base. The unmodified natural Cas12f parent has no mammalian-cell activity on its own.

References

1. Xu et al. (2021). Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing. Mol Cell - Xu 2021 CasMINI