1 research outputs found
Completely Free from PAM Limitations: Asymmetric RPA with CRISPR/Cas12a for Nucleic Acid Assays
Experimentally, Cas12a can recognize multiple protospacer
adjacent
motif (PAM) sequences and is not restricted to the “TTTN”.
However, the application of the CRISPR/Cas12a system is still limited
by the PAM for double-stranded DNA (dsDNA). Here, we developed asymmetric
RPA (Asy-RPA) to completely break the limitations of PAM. Asy-RPA
not only achieved efficient amplification but also converted dsDNA
to single-stranded DNA (ssDNA) without complicated steps. The ssDNA
products activated the trans-cleavage activity of
Cas12a, outputting signals. The application of Asy-RPA completely
freed Cas12a from the PAM, which can be more widely used in nucleic
acid detection, such as lumpy skin disease virus, with an actual detection
limit as low as 1.21 × 101 copies·μL–1. More importantly, Cas12a was intolerant to mutations
on ssDNA. This provided technical support for the detection and identification
of wild-type Mycobacterium tuberculosis (WT-TB) and rifampin-resistant mutant-type M. tuberculosis (MT-TB). The detection limit was as low as 1 fM for 1% mixed samples.
The detection and availability of different treatment options for
treatment-resistant and WT-TB were significant for the elimination
of TB. In summary, the platform consisting of Asy-RPA and CRISPR/Cas12a
was suitable for the detection of various viruses and bacteria and
was a boon for the detection of dsDNA without recognizable PAM