Two CRISPR/Cas12a-based methods for fast and accurate detection of single-base mutations

Current single-base mutation detection approaches are time-consuming, labor-intensive, and costly. This high-lights the critical need for speedy and accurate technology capable of detecting single-base alterations. Using clustered regularly interspaced short palindromic repeats/associated protein 12a (CRISPR/Cas12a), two fundamental approaches for getting 100% differentiation of single-base mutations have been established, by which fluorescence signals could be detected for variants but not for wild strains. The first method required both polymerase chain reaction (PCR) and CRISPR/Cas12a cleavage: By introducing a mismatched base at the 3 ' end of the primers and adjusting the PCR settings, the wild strain strand amplifications were completely blocked prior to CRISPR/Cas12a cleavage. The parameters for Method 1 (PCR + CRISPR/Cas12a) could be easily controlled and adjusted to attain a sensitivity of one copy (about 6 copies mu L-1). The second method included isothermal recombinase polymerase amplification (RPA) and CRISPR/Cas12a cleavage: By introducing an extra mismatched base adjacent to the single-base mutant site by RPA (IMAS-RPA), the RPA products from the wild strains were rendered incapable of triggering the cleavage activity of CRISPR/Cas12a. Method 2 (IMAS-RPA) was rapid and easy to implement (can be finished within 1 h). Because each method has its own set of advantages, the labo-ratory environment-appropriate methods can be selected independently. Both approaches are expected to aid in clinical diagnosis to some extent in the near future.

Automated summary

Current single-base mutation detection approaches are time-consuming, labor-intensive, and costly. Two methods based on CRISPR/Cas12a have been established for detecting single-base mutations with 100% differentiation, where fluorescence signals can be detected for variants but not for wild strains. Both methods have their own advantages, and they are expected to be applied in clinical diagnosis in the near future.