PAM-Independent Detection of Nucleic Acid Targets with CRISPR-Cas12a

The trans-cleavage activity of the type II class V CRISPR-Cas effector, Cas12a, has been widely utilized for molecular diagnostic applications through the detection of nucleic acid biomarkers. However, the requirement of a protospacer adjacent motif (PAM) site on the target double-stranded DNA (dsDNA) greatly limits its flexibility as a diagnostic tool. While Cas12a has been shown to optimally recognize a TTTV PAM motif, it can also tolerate non-canonical ‘C’ – containing PAMs such as CTTV, TCTV, TTCV, CCCV etc. to a lower extent. Nevertheless, Cas12a is not able to efficiently target substrates with ‘A’ and ‘G’ containing PAMs. Cas12a requires the presence of a PAM site only for the recognition and cleavage of dsDNA targets but not single-stranded DNA (ssDNA). In this work, we have developed a method for performing PAM-independent detection of nucleic acid substrates with LbCas12a. We achieved this by separating the dsDNA molecules into 2 separate ssDNA strands by subjecting them to a high temperature and high pH environment. We then detected each separated ssDNA strand with Cas12a in a PAM-independent manner. We showed that the isoelectric point of Cas12a plays an important role in predicting its activity at different pH. We also demonstrated that LbCas12a can function at a significantly higher pH environment as compared to other Cas12a orthologs. We utilized this characteristic of LbCas12a to develop a method named PAM-independent detection of nucleic acids with CRISPR-Cas12a or PICNIC. By coupling PICNIC with Reverse Transcriptase - Recombinase Polymerase Amplification (RT-RPA) we can achieve sensitive and specific detection of important nucleic acid targets, without the constraint of the PAM sequence. We believe PICNIC will greatly expand the targeting capabilities of Cas12a effectors and allow for efficient targeting of even those sequences that are not flanked by a PAM motif.