An engineered hypercompact CRISPR-Cas12f system with boosted gene-editing activity

Nat Chem Biol. 2023 Nov;19(11):1384-1393. doi: 10.1038/s41589-023-01380-9. Epub 2023 Jul 3.


Compact CRISPR-Cas systems offer versatile treatment options for genetic disorders, but their application is often limited by modest gene-editing activity. Here we present enAsCas12f, an engineered RNA-guided DNA endonuclease up to 11.3-fold more potent than its parent protein, AsCas12f, and one-third of the size of SpCas9. enAsCas12f shows higher DNA cleavage activity than wild-type AsCas12f in vitro and functions broadly in human cells, delivering up to 69.8% insertions and deletions at user-specified genomic loci. Minimal off-target editing is observed with enAsCas12f, suggesting that boosted on-target activity does not impair genome-wide specificity. We determine the cryo-electron microscopy (cryo-EM) structure of the AsCas12f-sgRNA-DNA complex at a resolution of 2.9 Å, which reveals dimerization-mediated substrate recognition and cleavage. Structure-guided single guide RNA (sgRNA) engineering leads to sgRNA-v2, which is 33% shorter than the full-length sgRNA, but with on par activity. Together, the engineered hypercompact AsCas12f system enables robust and faithful gene editing in mammalian cells.

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics
  • Cryoelectron Microscopy
  • DNA / chemistry
  • Gene Editing*
  • Humans
  • Mammals / genetics
  • RNA, Guide, CRISPR-Cas Systems*


  • RNA, Guide, CRISPR-Cas Systems
  • DNA