NIH-Funded UT Austin Team Engineers Mini-CRISPR Al3Cas12f with 90% Editing Efficiency for AAV Delivery

Researchers at the University of Texas at Austin, funded by NIH's National Institute of General Medical Sciences (NIGMS), engineer Al3Cas12f RKK — a compact CRISPR-Cas12f enzyme small enough to be packaged into adeno-associated virus (AAV) vectors, the leading targeted in-body delivery system for gene therapy. Using structural imaging and machine learning analysis, the team discovered that Al3Cas12f forms an unusually stable protein-RNA-DNA complex, then used directed evolution to engineer a variant (RKK) that boosts editing efficiency from less than 10% to more than 80% across tested genomic targets, reaching 90% at high-efficiency loci in human cells. The breakthrough addresses a fundamental bottleneck in CRISPR therapeutics: standard SpCas9 (4.2 kb) is too large for AAV packaging (cargo limit ~4.7 kb), forcing reliance on ex vivo cell engineering or LNP delivery limited to liver. A compact, high-efficiency Cas12 system small enough for AAV could enable direct systemic in-body gene editing for neurological, muscular, cardiovascular, and other diseases not accessible via ex vivo or hepatic LNP approaches. The work was published April 13, 2026 in Nature Structural & Molecular Biology.