Real‐time observation of flexible domain movements in CRISPR–Cas9

Abstract

The CRISPR‐associated protein Cas9 is widely used for genome editing because it cleaves target DNA through the assistance of a single‐guide RNA (sgRNA). Structural studies have revealed the multi‐domain architecture of Cas9 and suggested sequential domain movements of Cas9 upon binding to the sgRNA and the target DNA. These studies also hinted at the flexibility between domains; however, it remains unclear whether these flexible movements occur in solution. Here, we directly observed dynamic fluctuations of multiple Cas9 domains, using single‐molecule FRET. We found that the flexible domain movements allow Cas9 to adopt transient conformations beyond those captured in the crystal structures. Importantly, the HNH nuclease domain only accessed the DNA cleavage position during such flexible movements, suggesting the importance of this flexibility in the DNA cleavage process. Our FRET data also revealed the conformational flexibility of apo‐Cas9, which may play a role in the assembly with the sgRNA. Collectively, our results highlight the potential role of domain fluctuations in driving Cas9‐catalyzed DNA cleavage.

Implementation of single molecule FRET for visualizing intramolecular movement in CRISPR-Cas9

“…However, high anisotropy is not necessarily undesirable, because conformational changes coupled with not the distance but with the orientation of the dyes can be detected. In the case that low anisotropy is preferred, another combination of fluorescent dyes with long fluorescence lifetimes is recommended, because Cyanine dye 3 and Cyanine dye 5 show relatively short lifetimes (< 1 ns) 23 .…”

“…Note that the appropriate reagents for passivation depend on the observed bio-molecules and the surface conditions. For instance, casein is necessary for the single-molecule observation of microtubule-based motor movement 35,36 , but not in the case of Cas9 conformational changes 23, 37, 38 . It is always necessary to confirm that the observed protein has anchored on the surface via the intended linker.…”

“…Plot the transition density plot (TDP) using a graph visualization tool such as the matplotlib library for Python 32 to visualize the relationship between successive transitions in the states based on FRET efficiency 23 (Fig. 8).…”

“…smFRET can monitor the dynamics of two fluorescent dyes (donor and acceptor) labeling a target molecule with sub-nanometer accuracy and millisecond time resolution 22 . We have applied this system to reveal the flexible and reversible movements of the HNH domain in the DNA-sgRNA-Cas9 ternary complex 23 . The HNH domain was positioned at the DNA cleavage point only during flexible movements, suggesting the importance of domain flexibility in the Cas9 catalytic process.…”

Francisella novicidaCas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing

“…Interestingly, engineered highly specific Cas9 variants that are able to prevent cleavage at off targets containing PAM distal mismatches adopt a conformational structure that renders the HNH DNA cleavage domain inactive (Chen et al, 2017). Recent biophysical studies using single molecule fluorescence resonance energy transfer (smFRET) have revealed a highly dynamic conformation of spCas9 where allosteric interactions between the PAM distal end and the HNH domain of the Cas9 enzyme renders a cleavage-impaired conformationally 'closed' configuration upon encountering mismatches close to the 5' end of the sgRNA (Osuka et al, 2018;Yang et al, 2018). To dissect if a greater stringency of target recognition could be achieved in case of FnCas9 by increasing the number of mismatches at the 5' end of the substrate, we selected two wellstudied loci EMX1 and VEGFA3, amplified their genomic off targets with 2 and 3 mismatches at the non-seed region (PAM distal end) and interrogated the in vitro cleavage efficiency of FnCas9.…”