Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems

Ryan T. Leenay, Kenneth R. Maksimchuk, Rebecca A. Slotkowski, Roma N. Agrawal, Ahmed A. Gomaa, Alexandra E. Briner, Rodolphe Barrangou, Chase L. Beisel

Research output: Contribution to journalArticle

107 Scopus citations

Abstract

CRISPR-Cas adaptive immune systems in prokaryotes boast a diversity of protein families and mechanisms of action, where most systems rely on protospacer-adjacent motifs (PAMs) for DNA target recognition. Here, we developed an in vivo, positive, and tunable screen termed PAM-SCANR (PAM screen achieved by NOT-gate repression) to elucidate functional PAMs as well as an interactive visualization scheme termed the PAM wheel to convey individual PAM sequences and their activities. PAM-SCANR and the PAM wheel identified known functional PAMs while revealing complex sequence-activity landscapes for the Bacillus halodurans I-C (Cascade), Escherichia coli I-E (Cascade), Streptococcus thermophilus II-A CRISPR1 (Cas9), and Francisella novicida V-A (Cpf1) systems. The PAM wheel was also readily applicable to existing high-throughput screens and garnered insights into SpyCas9 and SauCas9 PAM diversity. These tools offer powerful means of elucidating and visualizing functional PAMs toward accelerating our ability to understand and exploit the multitude of CRISPR-Cas systems in nature.

Original languageEnglish (US)
Pages (from-to)137-147
Number of pages11
JournalMolecular cell
Volume62
Issue number1
DOIs
StatePublished - Apr 7 2016

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All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Cite this

Leenay, R. T., Maksimchuk, K. R., Slotkowski, R. A., Agrawal, R. N., Gomaa, A. A., Briner, A. E., Barrangou, R., & Beisel, C. L. (2016). Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems. Molecular cell, 62(1), 137-147. https://doi.org/10.1016/j.molcel.2016.02.031