Summary

    By deliberately introducing point mutations in Cas, our team created the Mut-4 mutant, which shares the same enzymatic activity as the mutant protein created by Doudna's team. Meanwhile, we used the hairpin-structured crRNA and achieved the optimal Cas-crRNA combination by tailoring the length of the hairpin structure to enhance the detection efficiency of the CRISPR-Cas system for single-base mismatch targets in nucleic acid detection applications. Our assay system outperformed traditional systems in terms of single-base mismatch target identification, which offers a better method for using the CRISPR-Cas system to detect nucleic acids. At the beginning of the research, we analysed the changes of six mutants in the optimized system, and analyze the changes in the skeleton carbon atom of the protein and the central structure of the entire protein through molecular dynamics simulation, which provided solid theoretical basis for our experiment. In order to broaden the CRISPR-Cas system's utility in nucleic acid detection, we want to develop a CRISPR-Cas system that can be used to detect small DNA mutations.