PT  - JOURNAL ARTICLE
AU  - Digvijay Singh
AU  - Samuel H. Sternberg
AU  - Jingyi Fei
AU  - Jennifer A. Doudna
AU  - Taekjip Ha
TI  - Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9
AID  - 10.1101/048371
DP  - 2016 Jan 01
TA  - bioRxiv
PG  - 048371
4099  - http://biorxiv.org/content/early/2016/04/12/048371.short
4100  - http://biorxiv.org/content/early/2016/04/12/048371.full
AB  - 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.