In Silico Processing of the Complete CRISPR-Cas Spacer Space for Identification of PAM Sequences

Despite extensive exploration of the diversity of CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR associated) systems, biological applications have been mostly confined to Class 2 systems, specifically the Cas9 and Cas12 (formerly Cpf1) single effector proteins. A key limitation of exploring and utilizing other CRISPR-Cas systems with unique functionalities, particularly Class I types and their multi-protein effector complex, is the knowledge of the system's protospacer adjacent motif (PAM) sequence identity. In this work, the authors developed a systematic pipeline, named CASPERpam, that enables a comprehensive assessment of the PAM sequences of all the available CRISPR-Cas systems in the NCBI database of bacterial genomes. The CASPERpam analysis reveals that within the 30 389 assemblies previously screened for CRISPR arrays, there exists 26 364 spacers that match somewhere in the viral, bacterial, and plasmid databases of NCBI, using the constraints of 95% sequence identity and 95% sequence coverage for blast hits. When grouping these results by species, the authors identified putative PAM sequences for 1049 among 1493 unique species. The remaining species either have insufficient data or an undetermined result from the analysis. Finally, the authors assigned a confidence score to each species' PAM prediction and generate categories that largely cover the revealed diversity of PAM motifs, providing a baseline for further experimental studies including PAM assays. The authors envision CASPERpam is a useful bioinformatic tool for understanding and harnessing the diversity of CRISPR-Cas systems.