title: In Vitro Reconstitution and Crystallization of Cas9 Endonuclease Bound to a Guide RNA and a DNA Target.
abstract: The programmable RNA-guided DNA cleavage activity of the bacterial CRISPR-associated endonuclease Cas9 is the basis of genome editing applications in numerous model organisms and cell types. In a binary complex with a dual crRNA:tracrRNA guide or single-molecule guide RNA, Cas9 targets double-stranded DNAs harboring sequences complementary to a 20-nucleotide segment in the guide RNA. Recent structural studies of the enzyme have uncovered the molecular mechanism of RNA-guided DNA recognition. Here, we provide protocols for electrophoretic mobility shift and fluorescence-detection size exclusion chromatography assays used to probe DNA binding by Cas9 that allowed us to reconstitute and crystallize the enzyme in a ternary complex with a guide RNA and a bona fide target DNA. The procedures can be used for further mechanistic investigations of the Cas9 endonuclease family and are potentially applicable to other multicomponent protein-nucleic acid complexes.
authors.affiliation: Department of Biochemistry, University of Zurich, Zurich, Switzerland.
authors.forename: Carolin
authors.initials: C
authors.lastname: Anders
authors.affiliation: Department of Biochemistry, University of Zurich, Zurich, Switzerland.
authors.forename: Ole
authors.initials: O
authors.lastname: Niewoehner
authors.affiliation: Department of Biochemistry, University of Zurich, Zurich, Switzerland. Electronic address: jinek@bioc.uzh.ch.
authors.forename: Martin
authors.initials: M
authors.lastname: Jinek
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D064130:CRISPR-Associated Proteins / Q000737:chemistry* / Q000235:genetics; D002850:Chromatography, Gel; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D003460:Crystallization; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D053837:DNA Cleavage; D024202:Electrophoretic Mobility Shift Assay; D004720:Endonucleases / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry* / Q000235:genetics; D013297:Streptococcus pyogenes / Q000737:chemistry / Q000201:enzymology
chemical_list: D064130:CRISPR-Associated Proteins; D012329:RNA, Bacterial; D000094704:RNA, Guide, CRISPR-Cas Systems; D004247:DNA; D004720:Endonucleases
keywords: CRISPR-Cas; Cas9; Double-strand DNA break; EMSA; Endonuclease; Fluorescence detection; Genome editing; HPLC; Macromolecular complexes; Protein–RNA interactions