Deciphering, Communicating, and Engineering the CRISPR PAM

• The PAM is an essential feature for target recognition.
• PAMs are associated with all characterized types of CRISPR–Cas systems.
• Multiple methods are available for PAM determination and visualization.
• Cas proteins can be engineered to alter, tighten, or relax PAM recognition
• We propose a standard means of orienting the PAM for all CRISPR–Cas types

Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their flanking CRISPR-associated (cas) genes make up RNA-guided, adaptive immune systems in prokaryotes whose effector proteins have become powerful tools for basic research and biotechnology. While the Cas effector proteins are remarkably diverse, they commonly rely on protospacer-adjacent motifs (PAMs) as the first step in target recognition. PAM sequences are known to vary considerably between systems and have proven to be difficult to predict, spurring the need for new tools to rapidly identify and communicate these sequences. Recent advances have also shown that Cas proteins can be engineered to alter PAM recognition, opening new opportunities to develop CRISPR-based tools with enhanced targeting capabilities. In this review, we discuss the properties of the CRISPR PAM and the emerging tools for determining, visualizing, and engineering PAM recognition. We also propose a standard means of orienting the PAM to simplify how its location and sequence are communicated.

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