ArticleMethods in enzymology2015
In Vitro Reconstitution and Crystallization of Cas9 Endonuclease Bound to a Guide RNA and a DNA Target.
Article in Methods in enzymology, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed, 31 citations in OpenAlex.
- Article
- Loss of circadian rhythmicity iniScience · 2022Article
- Article
- Multiplex CRISPR-Cas9 mutagenesis of the phytochrome gene family in Physcomitrium (Physcomitrella) patens.Plant molecular biology · 2021Article
- Article
- Targeted DNA transposition in vitro using a dCas9-transposase fusion protein.Nucleic acids research · 2019Article
- Enhanced Bacterial Immunity and Mammalian Genome Editing via RNA-Polymerase-Mediated Dislodging of Cas9 from Double-Strand DNA Breaks.Molecular cell · 2018Article
- Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity.Nature communications · 2018Article
- Bacteriophage DNA glucosylation impairs target DNA binding by type I and II but not by type V CRISPR-Cas effector complexes.Nucleic acids research · 2018Article
- Sequence-Directed Covalent Protein-DNA Linkages in a Single Step Using HUH-Tags.Journal of the American Chemical Society · 2017Article
- Cellular Reprogramming, Genome Editing, and Alternative CRISPR Cas9 Technologies for Precise Gene Therapy of Duchenne Muscular Dystrophy.Stem cells international · 2017Review
- The big bang of genome editing technology: development and application of the CRISPR/Cas9 system in disease animal models.Dong wu xue yan jiu = Zoological research · 2016Review
- Structural Plasticity of PAM Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9.Molecular cell · 2016Article
- Applications of CRISPR technologies in research and beyond.Nature biotechnology · 2016Article
Corrections and comments
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
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.
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