CRISPR-Cas12a biosensing via transcription of crRNA from PCR or LAMP products for pathogen detection

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 12a (Cas12a) is an RNA-guided nuclease that has been adapted for nucleic acid-based molecular diagnostics. However, the widespread adoption of Cas12-based molecular diagnostics has been limited by dependency of a protospacer adjacent motif (PAM) sequence within the target double-stranded DNA (dsDNA) required to activate Cas12a. To eliminate PAM dependency, we explored a noncanonical activation mode by designing tailed primers for PCR to generate crRNA from amplified target dsDNA products via transcription. The transcribed crRNA, along with a dsDNA activator, then activates Cas12a. We validated this method and named it PCR followed by Transcription And CRISPR-Cas12a (PCR-TRAC). We then developed loop-mediated isothermal amplification (LAMP)-TRAC using tailed primers. Both methods detected Mycobacterium tuberculosis genomic DNA extracted from clinical samples. LAMP-TRAC was more sensitive and faster than PCR-TRAC, detecting as little as four copies/μL of M. tuberculosis genomic DNA within 1 h. We envision that our CRISPR-Cas12-based diagnostic approach could be expanded to become a universal platform for identifying various other nucleic acid targets.