(27av) 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 achieve this by separating the dsDNA molecules into 2 separate ssDNA strands by subjecting them to a high temperature and high pH environment. We then detect each separated ssDNA strand with Cas12a in a PAM-independent manner. We show that the isoelectric point of Cas12a plays an important role in predicting its activity at different pH and demonstrate that LbCas12a can function at a significantly higher pH environment as compared to other Cas12a orthologs. We utilize 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 PAM sequence. We apply PICNIC for the detection of clinically relevant non-PAM containing targets within human immunodeficiency virus (HIV) and Hepatitis C Virus (HCV). 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.