Engineered CRISPR RNA improves the RNA cleavage efficiency of hfCas13X

As the most compact variant in the Cas13 family, CRISPR‐Cas13X holds considerable promise for gene therapy applications. The development of high‐fidelity Cas13X (hfCas13X) mutants has enhanced the safety profile for in vivo applications. However, a notable reduction in on‐target cleavage efficiency accompanies the diminished collateral cleavage activity in hfCas13X. In this study, we obtained two engineered crRNA mutants that notably enhance the on‐target cleavage efficiency of hfCas13X. Furthermore, we have identified a novel crRNA structure that consistently augments the on‐target cleavage efficiency of hfCas13X across various cellular environments, without significant enhancement of its collateral activity. These findings collectively enrich the gene‐editing toolkit, presenting a more effective hfCas13X system for future research and application.