CRISPR-Cas systems represent RNA-directed defense systems in prokaryotes that are revolutionizing genome engineering and gene regulation in bacteria through plants. While current efforts rely on a small handful of these systems, nature boasts thousands of CRISPR-Cas systems with unique properties, activities, specificities, and functions. The challenge is rapidly co-opting these uncharacterized systems for biotechnological purposes. Arguably the greatest barrier is elucidating the protospacer-adjacent motif (PAM) that is uniquely recognized by each CRISPR-Cas system and immediately flanks the target sequence. While the PAM is relatively short (2 – 5 nucleotides), the specific set of recognized PAM sequences varies widely between even related systems and has proven difficult to predict <i>a priori</i>. Here, we report a novel high-throughput screen called PAM-SCANR (PAM SCreen Associated with NOT-gated Repression) that relies on CRISPR-mediated repression of a transcriptional repressor of GFP in <i>E. coli</i>. As part of the screen, a functional PAM sequence leads to a fluorescent cell that can be isolated through fluorescence-activated cell sorting. Using this strategy, we identified functional PAM sequences for a range of previously characterized and uncharacterized CRISPR-Cas systems. These sequences in turn revealed that not all PAMs are equally functional and that systems recognize a much wider range of PAMs than previously realized. Overall, PAM-SCANR offers a rapid and quantitative screen that is poised to transport the vast number of uncharacterized CRISPR-Cas systems to the forefront of biotechnology.

Co-opting uncharacterized CRISPR-Cas systems as the next generation of biomolecular tools

251st National Meeting (2016)

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