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Related Experiment Video

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Real-Time, Two-Color Stimulated Raman Scattering Imaging of Mouse Brain for Tissue Diagnosis

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Time-resolved imaging-based CRISPRi screening.

Daniel Camsund¹, Michael J Lawson^2,3, Jimmy Larsson¹

¹Department of Cell and Molecular Biology, SciLifeLab, Uppsala University, Uppsala, Sweden.

|November 20, 2019

Summary

This summary is machine-generated.

This study introduces a new method combining live-cell imaging with in situ genotyping to link gene function to cell behavior. This approach allows researchers to screen thousands of gene knockdowns, advancing our understanding of cell cycle control in Escherichia coli.

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Area of Science:

Microbiology
Genomics
Cell Biology

Background:

Connecting genotype to phenotype is crucial but challenging due to limitations in live-cell microscopy and genomic engineering.
Existing methods struggle to bridge the gap between observing live cells and analyzing large-scale genetic modifications.

Purpose of the Study:

To develop and validate a novel technique for in situ genotyping after live-cell microscopy.
To investigate the impact of 235 CRISPR interference knockdowns on Escherichia coli cell cycle coordination.

Main Methods:

Utilizing a microfluidic device for time-lapse imaging of Escherichia coli strains.
Implementing in situ genotyping to link observed phenotypes to specific genetic perturbations.
Monitoring replication fork progression over multiple cell cycles (>500 per knockdown).

Main Results:

Successfully mapped phenotype distributions to specific gene knockdowns.
Identified genes critical for cell cycle control in Escherichia coli.
Characterized single-cell distributions of growth rates, division sizes, and replication initiation volumes.

Conclusions:

The developed technology overcomes limitations in connecting genotypic variation to cellular phenotypes.
This method enables genome-scale screening for live-cell microscopy-based assays.
Provides a powerful tool for dissecting complex biological processes at the single-cell level.