Live Cell Interrogation of dCas9:sgRNA Dynamics Off and At the Target

Although the CRISPR‐Cas9 system has been widely used for genome editing, little is known about its dynamics in live cells such as the kinetics of sgRNA:Cas9 assembly and DNA recognition. By labeling dCas9 (nuclease‐dead Cas9) using a fluorescent protein and sgRNAs with GFP‐like RNA aptamers (Broccoli), and employing a single genomic locus on human chromosome 3 as the target, we monitored the assembly of sgRNA:dCas9 and tracked its nuclear roaming and docking on the target. We generated stable cell lines with tunable dCas9 expression and constitutive sgRNA transcription. In the absence of dCas9 expression the sgRNA was so unstable as to not be detectable. In contrast, after dCas9 induction the sgRNA displayed two kinetic components, one with a half‐life of ca. 15 min. (likely sgRNA:dCas9 complexes roaming the nucleoplasm) and another more long‐lived (likely bound to both target and non‐target DNA). Fluorescence Recovery After Photobleaching (FRAP) revealed that sgRNA:dCas9 association with the target had an average residence time of 100 min. and that mismatches in the sgRNA seed significantly reduced the target residence time. A compelling correlation was observed between the target residence times of these sgRNA seed mutations and the cleavage activity observed for these same mutations in studies of others with nuclease‐active Cas9, suggesting that our tracking of dCa9 dynamics at the target reflects a basic thermodynamic feature of the loading step it shares with nuclease‐active Cas9. Our in vivo measurements, in combination with existing data from in vitro studies, should facilitate the design of efficient and precise CRISPR‐Cas9 applications in various cellular and therapeutic settings.