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Structural basis for the promiscuous PAM recognition by Corynebacterium diphtheriae Cas9
Seiichi Hirano1, Omar O Abudayyeh2,3, Jonathan S Gootenberg2,3
1Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
Nature Communications
|May 1, 2019
Summary
Corynebacterium diphtheriae Cas9 (CdCas9) uses unique interactions to recognize its NNRHHHY PAM sequence. This structural insight into CdCas9 function advances understanding of CRISPR-Cas9 genome editing tools.
Area of Science:
- Molecular Biology
- Structural Biology
- Genetics
Background:
- Cas9 nucleases are RNA-guided enzymes that cleave DNA at specific target sites adjacent to a protospacer adjacent motif (PAM).
- Different Cas9 orthologs exhibit diverse PAM recognition specificities, influencing their applications in genome editing.
- The mechanism by which Corynebacterium diphtheriae Cas9 (CdCas9) recognizes its promiscuous NNRHHHY PAM is not well understood.
Purpose of the Study:
- To elucidate the molecular mechanism of CdCas9 PAM recognition.
- To determine the structural basis for CdCas9's interaction with its target DNA and guide RNA.
- To provide insights into the engineering of CRISPR-Cas9 systems.
Main Methods:
- X-ray crystallography was employed to determine the structure of CdCas9 complexed with guide RNA and target DNA.
- Biochemical assays were used to assess CdCas9 DNA cleavage activity.
- Structural analysis focused on identifying key interactions between CdCas9, guide RNA, target DNA, and the PAM sequence.
Main Results:
- The crystal structure of CdCas9 bound to guide RNA and target DNA was determined at 2.9 Å resolution.
- CdCas9 recognizes the NNRHHHY PAM through a combination of van der Waals interactions and base-specific hydrogen bonds.
- CdCas9 demonstrated robust DNA cleavage activity, with optimal performance observed using 22-nucleotide guide RNAs.
Conclusions:
- The study reveals the unique structural mechanism underlying CdCas9's promiscuous NNRHHHY PAM recognition.
- Findings highlight the significant mechanistic diversity among Cas9 orthologs in PAM recognition.
- This structural and functional understanding of CdCas9 provides a foundation for developing improved CRISPR-Cas9 genome editing technologies.
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