Structure-Guided design of Cas12a variants improves detection of nucleic acids.

ABSTRACT: CRISPR-Cas12a holds promising potential for pathogen detection. However, its performance is not optimal when combined with isothermal amplification. Hence, we engineered a mutant of LbCas12a (K595A) with reduced cis-cleavage activity, to minimize interference with isothermal amplification. Compared to wild-type Cas12a, the K595A mutant exhibited a 2-3 times faster reaction speed and a 1,000-10,000 times increase in sensitivity in a one-pot reaction. We applied this mutant for detection of African Swine Fever Virus (ASFV). This K595A mutant successfully detected all 30 ASFV samples within 20 minutes. Our study suggests a universal approach to improve the performance of Cas12a for pathogen detection.

SUBMITTER: Tong X 

PROVIDER: S-EPMC11889556 | biostudies-literature | 2025 Apr

REPOSITORIES: biostudies-literature