The molecular basis for recognition of 5'-NNNCC-3' PAM and its methylation state by Acidothermus cellulolyticus Cas9
Abstract
Acidothermus cellulolyticus CRISPR-Cas9 (AceCas9) is a thermophilic Type II-C enzyme that has potential genome editing applications in extreme environments. It cleaves DNA with a 5'-NNNCC-3' Protospacer Adjacent Motif (PAM) and is sensitive to its methylation status. To understand the molecular basis for the high specificity of AceCas9 for its PAM, we determined two crystal structures of AceCas9 lacking its HNH domain (AceCas9-ΔHNH) bound with a single guide RNA and DNA substrates, one with the correct and the other with an incorrect PAM. Three residues, Glu1044, Arg1088, Arg1091, form an intricate hydrogen bond network with the first cytosine and the two opposing guanine nucleotides to confer specificity. Methylation of the first but not the second cytosine base abolishes AceCas9 activity, consistent with the observed PAM recognition pattern. The high sensitivity of AceCas9 to the modified cytosine makes it a potential device for detecting epigenomic changes in genomes.
- Publication Date:
- Research Org.:
- Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS); Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source II (NSLS-II)
- Sponsoring Org.:
- National Institutes of Health (NIH); National Institute of General Medical Sciences (NIGMS); Office of Research Infrastructure Programs (ORIP); USDOE Office of Science (SC), Biological and Environmental Research (BER)
- OSTI Identifier:
- 1763125
- Grant/Contract Number:
- R01 GM099604; S10 RR029205; S10OD021527; AC02-06CH11357; SC0012704; P41GM111244; KP1605010
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Nature Communications
- Additional Journal Information:
- Journal Volume: 11; Journal Issue: 1; Journal ID: ISSN 2041-1723
- Publisher:
- Nature Publishing Group
- Country of Publication:
- United States
- Language:
- ENGLISH
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; CRISPR-Cas systems; DNA; DNA methylation; X-ray crystallography
Citation Formats
Das, Anuska, Hand, Travis H., Smith, Chardasia L., Wickline, Ethan, Zawrotny, Michael, and Li, Hong. The molecular basis for recognition of 5'-NNNCC-3' PAM and its methylation state by Acidothermus cellulolyticus Cas9. United States: N. p., 2020.
Web. doi:10.1038/s41467-020-20204-1.
Das, Anuska, Hand, Travis H., Smith, Chardasia L., Wickline, Ethan, Zawrotny, Michael, & Li, Hong. The molecular basis for recognition of 5'-NNNCC-3' PAM and its methylation state by Acidothermus cellulolyticus Cas9. United States. https://doi.org/10.1038/s41467-020-20204-1
Das, Anuska, Hand, Travis H., Smith, Chardasia L., Wickline, Ethan, Zawrotny, Michael, and Li, Hong. Fri .
"The molecular basis for recognition of 5'-NNNCC-3' PAM and its methylation state by Acidothermus cellulolyticus Cas9". United States. https://doi.org/10.1038/s41467-020-20204-1. https://www.osti.gov/servlets/purl/1763125.
@article{osti_1763125,
title = {The molecular basis for recognition of 5'-NNNCC-3' PAM and its methylation state by Acidothermus cellulolyticus Cas9},
author = {Das, Anuska and Hand, Travis H. and Smith, Chardasia L. and Wickline, Ethan and Zawrotny, Michael and Li, Hong},
abstractNote = {Acidothermus cellulolyticus CRISPR-Cas9 (AceCas9) is a thermophilic Type II-C enzyme that has potential genome editing applications in extreme environments. It cleaves DNA with a 5'-NNNCC-3' Protospacer Adjacent Motif (PAM) and is sensitive to its methylation status. To understand the molecular basis for the high specificity of AceCas9 for its PAM, we determined two crystal structures of AceCas9 lacking its HNH domain (AceCas9-ΔHNH) bound with a single guide RNA and DNA substrates, one with the correct and the other with an incorrect PAM. Three residues, Glu1044, Arg1088, Arg1091, form an intricate hydrogen bond network with the first cytosine and the two opposing guanine nucleotides to confer specificity. Methylation of the first but not the second cytosine base abolishes AceCas9 activity, consistent with the observed PAM recognition pattern. The high sensitivity of AceCas9 to the modified cytosine makes it a potential device for detecting epigenomic changes in genomes.},
doi = {10.1038/s41467-020-20204-1},
journal = {Nature Communications},
number = 1,
volume = 11,
place = {United States},
year = {Fri Dec 11 00:00:00 EST 2020},
month = {Fri Dec 11 00:00:00 EST 2020}
}
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