PAM-independent ultra-specific activation of CRISPR-Cas12a via sticky-end dsDNA

Although CRISPR-Cas12a [clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 12a] combining pre-amplification technology has the advantage of high sensitivity in biosensing, its generality and specificity are insufficient, which greatly restrains its application range. Here, we discovered a new targeting substrate for LbaCas12a (Lachnospiraceae bacterium Cas12a), namely double-stranded DNA (dsDNA) with a sticky-end region (PAM(-)SE(+) dsDNA). We discovered that CRISPR-Cas12a had special enzymatic properties for this substrate DNA, including the ability to recognize and cleave it without needing a protospacer adjacent motif (PAM) sequence and a high sensitivity to single-base mismatches in that substrate. Further mechanism studies revealed that guide RNA (gRNA) formed a triple-stranded flap structure with the substrate dsDNA. We also discovered the property of low-temperature activation of CRISPR-Cas12a and, by coupling with the unique DNA hybridization kinetics at low temperature, we constructed a complete workflow for low-abundance point mutation detection in real samples, which was fast, convenient and free of single-stranded DNA (ssDNA) transformation. The detection limits were 0.005-0.01% for synthesized strands and 0.01-0.05% for plasmid genomic DNA, and the mutation abundances provided by our system for 28 clinical samples were in accordance with next-generation sequencing results. We believe that our work not only reveals novel information about the target recognition mechanism of the CRISPR-Cas12a system, but also greatly broadens its application scenarios.