Strategy used to engineer inducible CNTROB knockout and validation of knockout by genotyping and centrosome immunofluorescence. (A) Strategy used to engineer iCNTROB KO in TRIM37Δ and USP28Δ RPE1 cells (i) and details of the CNTROB gRNA, indicating target site in the CNTROB locus (ii). Note that both cell lines have in situ mNG-tagged CEP192. To validate the efficacy of the knockout, tracking of indels by decomposition (TIDE) analysis (Brinkman et al., 2014) was conducted 4 d after induction with 1 µM doxycycline. In both cell lines, ∼80% indels were observed in the induced population. (B) Quantification of centrobin signal at centrosomes 4 d after induction of the knockout. Mean and 95% CI are plotted on top of measurements of individual centrosome pairs (the signal for both centrosomes in a cell was measured together); P value is from an unpaired t test. Experiment performed twice; data shown is from one experiment.