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Updated: Mar 14, 2026
CIRCLE-Seq for Interrogation of Off-Target Gene Editing
Published on: November 1, 2024
Dynamics of CRISPR-Cas9 genome interrogation in living cells
Spencer C Knight1, Liangqi Xie2, Wulan Deng3
1Department of Chemistry, University of California, Berkeley, CA, USA.
Abstract:
The RNA-guided CRISPR-associated protein Cas9 is used for genome editing, transcriptional modulation, and live-cell imaging. Cas9-guide RNA complexes recognize and cleave double-stranded DNA sequences on the basis of 20-nucleotide RNA-DNA complementarity, but the mechanism of target searching in mammalian cells is unknown. Here, we use single-particle tracking to visualize diffusion and chromatin binding of Cas9 in living cells. We show that three-dimensional diffusion dominates Cas9 searching in vivo, and off-target binding events are, on average, short-lived (<1 second). Searching is dependent on the local chromatin environment, with less sampling and slower movement within heterochromatin. These results reveal how the bacterial Cas9 protein interrogates mammalian genomes and navigates eukaryotic chromatin structure.
Insights
The CRISPR-associated protein Cas9 uses 3D diffusion to search for DNA targets in mammalian cells. Its movement and sampling are influenced by the local chromatin environment, revealing its genome interrogation mechanism.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The CRISPR-associated protein Cas9 (CRISPR-Cas9) is a powerful tool for genome editing, transcriptional modulation, and live-cell imaging.
- CRISPR-Cas9 complexes target double-stranded DNA via RNA-DNA complementarity but the in vivo search mechanism in mammalian cells remains unclear.
Purpose of the Study:
- To investigate the in vivo mechanism of target searching by Cas9 in mammalian cells.
- To visualize the diffusion and chromatin binding dynamics of Cas9 in living cells.
Main Methods:
- Single-particle tracking was employed to monitor the movement and chromatin interactions of Cas9 in live cells.
- Analysis focused on diffusion patterns and the duration of off-target binding events.
Main Results:
- Cas9 primarily utilizes three-dimensional diffusion for target searching within mammalian cells.
- Off-target binding events are transient, lasting less than one second on average.
- Cas9 movement and chromatin sampling are restricted in heterochromatin regions, indicating environmental dependence.
Conclusions:
- The study elucidates the in vivo genome interrogation strategy of bacterial Cas9 in eukaryotic cells.
- Cas9 navigates mammalian genomes by balancing diffusion with chromatin accessibility, influenced by local chromatin structure.
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