A cas12a protein mutant with high cleavage activity and application thereof

By mutating the amino acid sequence of the Cas12a protein, its nuclease activity and specificity were improved, solving the problem of insufficient sensitivity of the CRISPR/Cas12a system to single base mismatches and realizing high-sensitivity detection of SNP sites.

CN122256305APending Publication Date: 2026-06-23NAT HEALTH COMMISSION INST OF SCI & TECH
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
NAT HEALTH COMMISSION INST OF SCI & TECH
Filing Date
2026-02-12
Publication Date
2026-06-23

AI Technical Summary

Technical Problem

The CRISPR/Cas12a system has insufficient sensitivity to single base mismatches, especially in SNP detection, where it has limited ability to identify heterozygous samples and cannot effectively distinguish between homozygous and heterozygous sites, thus limiting its application in SNP detection.

Method used

By engineering the Cas12a protein, especially by mutating the amino acid sequences of LbCas12a, FnCas12a and GhCas12a, its nuclease activity and specificity are improved, and a nucleic acid detection protein with higher sensitivity and specificity is developed.

Benefits of technology

This technology enables highly sensitive detection of single-base mismatches, effectively distinguishing between homozygous and heterozygous SNP sites and enhancing the application potential of the CRISPR/Cas12a system in SNP detection.

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Abstract

The application belongs to the technical field of biology and specifically relates to a Cas12a protein mutant with high cleavage activity and application thereof. The Cas12a protein mutant has high trans-cleavage activity and has a good application prospect in nucleic acid detection. The Cas12a protein mutant changes the binding of the protein and crRNA by directional evolution of the RECII domain, improves the cleavage efficiency of the target strand in double-stranded DNA, and improves the detection sensitivity, and can distinguish single nucleotide polymorphism (SNP). The Cas12a protein mutant can hardly depend on PAM sequence recognition for SNP site detection, can distinguish SNP located between positions 1 to 17, and realizes high-sensitivity and high-precision molecular detection.
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