Apr 13, 2016

Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9

BioRxiv : the Preprint Server for Biology
Digvijay SinghJennifer A Doudna

Abstract

Binding specificity of Cas9-guide RNA complexes to DNA is important for genome engineering applications, but how mismatches influence target recognition and rejection kinetics is not well understood. We used single-molecule FRET to probe real-time interactions between Cas9-RNA and DNA targets. The bimolecular association rate is only weakly dependent on sequence, but the dissociation rate greatly increases from < 0.006 s-1 to > 2 s-1 upon introduction of mismatches proximal to the protospacer adjacent motif (PAM), demonstrating that mismatches encountered early during heteroduplex formation induce rapid rejection of off-target DNA. In contrast, PAM-distal mismatches up to 12 base pairs in length, which prevent DNA cleavage, still allow the formation of a stable complex (off-rate < 0.006 s-1), suggesting that extremely slow rejection could sequester Cas9-RNA, increasing the Cas9 expression level necessary for genome editing thereby aggravating off-target effects. We also observed at least two different bound FRET states that may represent distinct steps in target search and proofreading.

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Mentioned in this Paper

PAM Protocol
AN 1
Endonuclease
Complex (molecular entity)
Heteroduplex Formation
DNA Cleavage
Protein Domain
Recognition (Psychology)
Base Pairing
Plasmids

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