Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems
| Autor: | Kenneth R. Maksimchuk, Ryan T. Leenay, Rodolphe Barrangou, Rebecca A. Slotkowski, Alexandra E. Briner, Ahmed A. Gomaa, Roma N. Agrawal, Chase L. Beisel |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Protein family CRISPR-Associated Proteins Bacillus Computational biology Biology medicine.disease_cause Article Microbiology 03 medical and health sciences 0302 clinical medicine Escherichia coli medicine Streptococcus thermophilus CRISPR Francisella novicida Francisella Molecular Biology Cas9 Extramural Cell Biology High-Throughput Screening Assays Protein Structure Tertiary Protospacer adjacent motif 030104 developmental biology CRISPR-Cas Systems 030217 neurology & neurosurgery |
| Zdroj: | Molecular Cell. 62:137-147 |
| ISSN: | 1097-2765 |
| DOI: | 10.1016/j.molcel.2016.02.031 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|