The molecular basis for recognition of 5\u2032-NNNCC-3\u2032 PAM and its methylation state by Acidothermus cellulolyticus Cas9

Anuska Das,Travis H Hand,Chardasia L Smith,Michael Zawrotny,Hong Li,Ethan Wickline

doi:10.1038/s41467-020-20204-1

Anuska Das, Travis H Hand + Show 4 more

Open Access

https://doi.org/10.1038/s41467-020-20204-1

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Journal: Nature Communications	Publication Date: Dec 1, 2020
Citations: 17	License type: open-access

Affiliation: Florida State University

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.

Highlights

Acidothermus cellulolyticus CRISPR-Cas[9] (AceCas9) is a thermophilic Type II-C enzyme that has potential genome editing applications in extreme environments
We show that AceCas[9] contacts the 5′-NNNCC-3′ Protospacer Adjacent Motif (PAM) by forming a network of hydrogen bonds with both the cytosine and guanine bases
Our previous work showed that 5′-NNNCC-3′ is a functional PAM for AceCas[9] but did not explore other possible PAMs that may support AceCas[9] functions[29]

Summary

Introduction

Acidothermus cellulolyticus CRISPR-Cas[9] (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. A critical element of the DNA target recognized by Cas[9] is called protospacer adjacent motif (PAM). Whereas promiscuous PAM sequences lead to a greater targeting range, they are more inducive to potential offtargets, which could hinder safe therapeutic application of Cas[9]. Given the essentiality of the PAM, the molecular basis for its interaction with Cas[9] will benefit further development of Cas9-based technology.

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