Publication Date:
2014-08-01
Description:
The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA. Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5'-NGG-3' PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA-DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4176945/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉 〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4176945/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Anders, Carolin -- Niewoehner, Ole -- Duerst, Alessia -- Jinek, Martin -- 337284/European Research Council/International -- England -- Nature. 2014 Sep 25;513(7519):569-73. doi: 10.1038/nature13579. Epub 2014 Jul 27.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/25079318" target="_blank"〉PubMed〈/a〉
Keywords:
Arginine/genetics/metabolism
;
*Base Pairing
;
Base Sequence
;
CRISPR-Associated Proteins/*metabolism
;
Crystallography, X-Ray
;
DNA/*chemistry/genetics/*metabolism
;
Endonucleases/*metabolism
;
Models, Molecular
;
Nucleic Acid Denaturation
;
*Nucleotide Motifs
;
Protein Conformation
;
RNA, Guide/chemistry/genetics/metabolism
;
Streptococcus pyogenes/*enzymology
;
Substrate Specificity
Print ISSN:
0028-0836
Electronic ISSN:
1476-4687
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
,
Natural Sciences in General
,
Physics
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