Visualisation of dCas9 target search in vivo using an open-microscopy framework

Area of Science:

• Molecular Biology
• Genetics
• Biophysics

Background:

• CRISPR-Cas9 is a powerful genomic editing tool, but its target search kinetics and dependence on Cas9 concentration are not fully understood.
• Efficient CRISPR function relies on rapid screening of protospacer adjacent motifs (PAMs), with recent studies suggesting a 30 ms upper limit for this process.

Purpose of the Study:

• To quantitatively analyze the dynamic behavior of deactivated Cas9 (dCas9) between DNA-bound and freely-diffusing states.
• To determine the PAM screening frequency and binding event duration for dCas9 in a cellular context.
• To investigate the relationship between Cas9 expression levels, target DNA copy number, and cleavage efficiency.

Main Methods:

• Development of an open-microscopy framework called miCube.
• Application of Monte-Carlo diffusion distribution analysis (MC-DDA) to quantify dCas9 dynamics.
• Utilizing heterogeneous dCas9 expression to study copy number-dependent Cas9 cleavage.

Main Results:

• dCas9 spends 40% of its time screening PAMs in Lactococcus lactis, with an average binding event duration of 17 ± 4 ms.
• The study derived Cas9 cleavage rates based on cellular target-containing plasmid copy numbers.
• It was demonstrated that dCas9 does not bind irreversibly to target sites and can impede plasmid replication.

Conclusions:

• The quantitative insights into dCas9 kinetics provide a deeper understanding of CRISPR-Cas system efficiency.
• These findings are crucial for optimizing the design and application of CRISPR-based genome editing tools.
• The developed miCube framework and MC-DDA offer valuable methods for studying molecular interactions in vivo.