Many pathogens encounter severe crowding inside host tissues, yet we still do not know which bacterial genes let them stay alive when their cell envelopes are literally being squeezed. To close this gap we will combine a pooled CRISPR-interference (CRISPRi) library with our microfluidic confinement assay. Cells carrying thousands of different guide-RNAs will fill a narrow growth channel until high internal pressure develops; short-read Illumina sequencing of guide barcodes before and after confinement will then reveal which knock-downs hurt—or help—survival. Training on the sequencing workflow is essential for us to turn raw files into a ranked list of “pressure-tolerance” genes.
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