PAM-Independent Cas12a Detection of Specific LAMP Products by Targeting Amplicon Loops

A straightforward approach is suggested to selectively recognize specific products of loop-mediated isothermal amplification (LAMP) by Cas12a nuclease without a need for a protospacer adjacent motif (PAM) in the sequence of LAMP amplicons (LAMPlicons). The strategy is based on the presence of single-stranded DNA loops in LAMPlicons and the ability of Cas12a to be trans-activated via binding of guide RNA (gRNA) to single-stranded DNA in the absence of PAM. The approach feasibility is demonstrated on Clavibacter species – a bunch of bacterial plant pathogens causing harmful diseases of agriculturally important plants. In regard to Clavibacter species, the detection sensitivity of the developed PAM-independent LAMP/Cas12a system was determined by that of LAMP, while the overall detection selectivity was enhanced by the Cas12a analysis of LAMPlicons. It was shown that the LAMP/Cas12a detection system can be fine-tuned by carefully designing gRNA so to selectively distinguish C. sepedonicus among other Clavibacter species, based on single nucleotide substitutions in the targeted LAMPlicon loop. The suggested loop-based Cas12a analysis of LAMPlicons was compatible with a format of single test tube assay with the option of naked-eye detection. The findings broaden the palette of approaches to designing PAM-independent LAMP/Cas12a detection systems with a potential for on-site testing.