Fig. 1: Genome-wide pooled CRISPR screen identified KIRREL1 as a positive regulator of Hippo pathway.

a Schematic of FACS-based pooled CRISPR screen to identify positive regulators of Hippo pathway in HEK293A GTIIC-GFP^PEST Cas9 cells. b Frequency histograms of gRNAs identified in the high GFP^PEST population of HEK293A GTIIC-GFP^PEST pooled CRISPR screen. The RSA p-value is shown along with log2 fold change for each of the five gRNAs per gene for the indicated set of genes. gRNAs targeting the indicated genes are shown by the red lines. c Knockout of KIRREL1 by independent gRNAs enhances GTIIC-GFP^PEST reporter activity in FACS assay. HEK293A GTIIC-GFP^PEST Cas9 cells transduced with lentivirus expressing RFP and control or KIRREL1 gRNAs were mixed with parental cells at 1:3 ratio, co-cultured for 2 days, and subjected to FACS analysis. The signal of GTIIC-GFP^PEST in RFP-positive cells is plotted. d Knockout of KIRREL1 increases YAP target gene expression in HEK293A cells. HEK293A GTIIC-GFP^PEST cells expressing control or KIRREL1 gRNAs were plated at medium density (0.5 × 10⁶ cells/well) in 12-well plate and subjected for qRT-PCR analysis 48 h of post cell plating. The data represent mean ± SD, n = 4, error bars denote the SD between four biological replicates; Unpaired two-tailed t-test was used to determine the statistical significance ***p value < 0.001. e Knockout of KIRREL1 increases the protein level of TAZ and decreases YAP phosphorylation in HEK293A cells. Control and KIRREL1 knockout cells were plated at high (1 × 10⁶ cells/well), medium (0.5 × 10⁶ cells/well), or low (0.25 × 10⁶ cells/well) density in 12-well plate and subjected to western blot analysis 24 h of post cell plating. GAPDH was used as loading control. f Depletion of KIRREL1 using Dox-inducible shRNAs leads to increased GTIIC-GFP^PEST reporter activity. HEK293A-GTIIC-GFP^PEST cells stably expressing control or two independent Dox-inducible shRNAs targeting KIRREL1 and subjected to FACS analysis following 96 h of Dox treatment. g Depletion of by shRNA increases expression of YAP target genes in HEK293A cells. The data represent mean ± SD, n = 4, error bars denote the SD between four biological replicates; Unpaired two-tailed t-test was used to determine the statistical significance, ***p value < 0.001. Source data for Fig. 1d, e, g are provided as Source Data file.