Screens for nuclear size regulators. (a) Example images of the nuclear size screen. Cells (hTERT-RPE1 dCas9-KRAB-BFP PA-mCherry H2B-mGFP) were transduced with a CRISPRi sgRNA library and imaged under the GFP channel. Cells with nuclei >1,000 µm² were photoactivated, sorted, and analyzed by deep sequencing. Example images of nuclei, (GFP channel, green), before and after photoactivation (mCherry channel, red). Note that the example images were from experiments done with dual-activation setup as described in Fig. 3 c. Background cells with low fluorescence intensity in mCherry channel after photoactivation were true negative cells that were photoactivated with a shorter exposure time (200 ms). Scale bar: 100 µm. (b) Workflow of one replicate of the nuclear size screen. For each replicate, transduced cells were seeded into four glass-bottom imaging plates. Cells in each single imaging plate were imaged, analyzed, photoactivated, sorted, and sequenced separately, termed as separate runs. (c) Screening result of one replicate shown in volcano plot. The corresponding eFDR-η curve is shown in Fig. S3 b, and the other replicate is shown in Fig. S3 c. (d) Comparison between two replicates. (e) List of genes identified from two replicates. Red, hit; gray, non-hit. (f) Workflow of three screens, namely nuclear size screen, H2B-mGFP screen, and FSC screen. After transducing the sgRNA library, cells were split and prepared for either image analysis (nuclear size screen) or FACS sorting (H2B-mGFP screen and FSC screen). (g) Comparison of hits identified in FSC screen, H2B-mGFP screen, and nuclear size screen.