PAM-Scanr: An In Vivo Positive Screen to Elucidate Protospacer-Adjacent Motifs for Uncharacterized Crispr-Cas Systems

Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins (CRISPR-Cas) systems are programmable endonucleases that have recently emerged as a popular genomic engineering tool. However, of the thousands of CRISPR-Cas systems found in nature, scientists rely on only a handful. This is partly due to a lack of procedures available to characterize new system requirements. In type I and II systems, a protospacer-adjacent-motif (PAM) must also be present for the CRISPR-Cas to recognize the target sequence. Different CRISPR-Cas systems recognize different PAMs, so uncharacterized type I and II CRISPR-Cas systems must be tested for PAM recognition before they can be used. Currently, there are only two methods for discovering new PAM, but theses methods have limitations such as expense or incompatibility with type I CRISPR-Cas systems. To overcome these limitations, we have developed an in vivo positive screen to determine CRISPR-Cas PAMs called PAM-SCreen Acquired by NOT-gate Repression (PAM-SCANR).  In our assay, a synthetic reporter plasmid contains a PAM library upstream of a repressor targeting GFP. If the correct PAM is present, the cells will fluoresce. We have successfully applied our assay to determine PAMs for type I and type II CRISPR-Cas systems and can reveal biases in the PAM sequence. In addition, the assay showed that PAM specificity is a continuum rather than an all-or-nothing phenomenon. New type I and II CRISPR-Cas systems can be characterized with the PAM-SCANR, thereby broadening the available set of genomic engineering tools.