Figure 2.

CRISPR-Cas9-mediated loss-of-function screen for cell deformability. A) Illustration of the CRISPR-Cas9 and microfluidic chip screening strategy. Cells were transduced with a lentiCRISPR kinase library and sorted by deformability in an MS-Chip. The flexible cells were allowed to flow out of the MS-Chip (the output) and collected for parallel sequencing together with the untreated whole cells (the input). Cell deformation was visualized by microscopy as a cell passed through a microconstriction (scale bar: 10 μm). B) Western blot analysis of nontransduced MDA-MB-231 cells and MDA-MB-231 cells transduced with a doxycycline-inducible FLAG-Cas9 construct upon doxycycline induction. Actin was used as the loading control. C) Statistical analysis of the on-chip transport distance (at a flow rate of 25 μLmin⁻¹) versus cell diameter for the CRISPR kinase-KO cells. Cells expressing FLAG-Cas9 only were used as the control. The red and blue circles indicate 80% confidence intervals from the means. The means are depicted by solid circles. D) MA plot of mean normalized counts versus log₂(fold change) for the output and input sgRNAs. The arrow indicates the top sgRNA hits with log₂(fold change)> 7 and an adjusted p value of <0.001.