Loop engineering of AtCas9 for effective and broad genome editing

Efficient genome editing in mammalian cells is essential for CRISPR-based therapeutics. While extensive efforts focused on combining multiple beneficial point mutations to enhance Cas9 activity, the potential of engineering surface-exposed loops remains largely underexplored. Here, we present loop engineering as a streamlined strategy to enhance Cas9 performance. Substituting loops of thermophilic AtCas9 with counterparts from mesophilic Nme1Cas9 generated the AtCas9-Z7 variant, which significantly improves nuclease and base editing efficiency. Biochemical assays showed that Mg²⁺ promotes RNP-DNA interactions, and Z7 maintains high binding affinity under magnesium-limiting condition, a common constraint for Cas9 activity in mammalian cells. Molecular dynamics simulations revealed that Z7 adopts a stable, compact conformation. Importantly, loop engineering can be combined with structure-guided point mutations to further boost activity. The resulting Z7-E78-ABE variant not only achieved a 5.76-fold increase compared to WT AtCas9 and expanded PAM recognition, while enabling editing in primary human T cells, which was not observed with WT AtCas9. Extending this strategy, loop transplantation into GeoCas9 and ThermoCas9 boosted editing efficiency by a median of 14.50-fold and 7.37-fold, respectively, at non-canonical PAMs. Collectively, these results establish loop engineering as a rational and modular approach for Cas9 optimization with therapeutic potential.