Optimization of CRISPR/Cas12f1 guide RNAs using AlphaFold 3 for enhanced nucleic acid detection

Abstract

The CRISPR/Cas12f1 system, known for its unique trans-cleavage activity, has emerged as a promising tool for nucleic acid detection. However, the optimization of guide RNAs in Cas12f1 remains a critical challenge. This study employs AlphaFold 3, a cutting-edge AI-driven structural prediction tool, to increase the trans-cleavage efficiency of Cas12f1 ribonucleoprotein (RNP) complexes through strategic guide RNA modifications. Initial structural simulations of wild-type tracrRNA and crRNA sequences revealed unexpected pairing within tracrRNA, which disrupted its interaction with crRNA, thereby reducing Cas12f1 activity. To address this, we truncated the 3′ end of tracrRNA, eliminating disruptive pairing and significantly improving the system’s trans-cleavage activity. To this end, we designed a single guide RNA (sgRNA) by linking the optimized tracrRNA and crRNA. Structural simulations confirmed that the sgRNA formed stable duplexes between tracrRNA and crRNA parts similar to those in the optimized tracrRNA-crRNA complex. Compared with the truncated tracrRNA system, the sgRNA further increased the trans-cleavage activity of the Cas12f1 RNP, with improved sensitivity compared to the truncated tracrRNA system. These findings underscore the potential of structure-based guide RNA optimization via AlphaFold 3 to advance the development of more efficient CRISPR/Cas12f1 biosensing systems for nucleic acid detection. This approach offers a novel and effective strategy for improving the performance of CRISPR-based technologies in various diagnostic applications.