Abstract
CRISPR-Cas systems have transformed genome editing, yet the commonly used Streptococcus pyogenes Cas9 (SpCas9) is limited by off-target effects and chromosomal instability. Here, we characterize AaCas12bMAX, an engineered Alicyclobacillus acidiphilus Cas12b variant, as a high-precision editing platform optimized for tumor-infiltrating lymphocyte (TIL) therapy. Using an FDA-compliant safety assessment framework, we systemically compared AaCas12bMAX- and SpCas9-edited TIL products in terms of on-target efficiency, genome-wide off-target activity, and structural variant (SV) formation. AaCas12bMAX achieved >80% on-target editing efficiency with undetectable off-target events and a 3.3-fold reduction in SVs relative to SpCas9. Mechanistic studies revealed different DNA repair kinetics in AaCas12bMAX-edited cells, reducing sustained DNA damage responses and chromosomal instability. Structural modeling suggested a more stable enzyme-sgRNA-DNA ternary complex, enabling stringent PAM specificity and minimal mismatch tolerance. Functionally, AaCas12bMAX-edited TILs exhibited superior therapeutic potential, including enhanced cellular fitness, a 2-fold increase in expansion capacity, and enrichment of stem-like tumor-reactive CD39-CD69-CD8+ subsets. Together, these results establish AaCas12bMAX as a robust, clinically translatable platform that improves the safety and functional limitations of SpCas9, enabling the development of next-generation T cell therapies.