In Vitro Reconstitution and Crystallization of Cas9 Endonuclease Bound to a Guide RNA and a DNA Target

Methods Enzymol. 2015:558:515-537. doi: 10.1016/bs.mie.2015.02.008. Epub 2015 Mar 12.


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.

Keywords: CRISPR-Cas; Cas9; Double-strand DNA break; EMSA; Endonuclease; Fluorescence detection; Genome editing; HPLC; Macromolecular complexes; Protein–RNA interactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Binding Sites
  • CRISPR-Associated Proteins / chemistry*
  • CRISPR-Associated Proteins / genetics
  • Chromatography, Gel
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • Crystallization
  • DNA / chemistry*
  • DNA / genetics
  • DNA Cleavage
  • Electrophoretic Mobility Shift Assay
  • Endonucleases / chemistry*
  • Endonucleases / genetics
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Binding
  • RNA, Bacterial / chemistry*
  • RNA, Bacterial / genetics
  • RNA, Guide, Kinetoplastida / chemistry*
  • RNA, Guide, Kinetoplastida / genetics
  • Streptococcus pyogenes / chemistry
  • Streptococcus pyogenes / enzymology


  • CRISPR-Associated Proteins
  • RNA, Bacterial
  • RNA, Guide
  • DNA
  • Endonucleases