Gene silencing of cyclin B1 and cyclin B2. (A) Conditional gene silencing strategy for mitotic cyclin B. CRISPR-Cas9 was used to disrupt the loci of both endogenous cyclin B1 and cyclin B2. The cDNA of cyclin B1 was tagged with mAID and put inside a Sleeping Beauty transposon cassette for genome delivery to rescue the KO effects. Silent mutations were introduced into ^mAIDcyclin B1 to confer resistance to the CRISPR-Cas9. In the presence of Dox, transcription of ^mAIDcyclin B1 is inhibited by blocking the tetracycline-controlled transcriptional activator (tTA) from binding to the TRE in the promoter. The addition of IAA triggers the degradation of residual ^mAIDcyclin B1 in cells expressing the F-box protein TIR1. ITR: inverted terminal repeat. (B) Gene silencing of cyclin B1 and/or cyclin B2. HeLa cells were engineered to stably express ^mAIDcyclin B1, tTA, and TIR1. CRISPR–Cas9 was used to disrupt cyclin B1 (in ^mAIDB1^KOB1) or both cyclin B1 and B2 (in ^mAIDB1^KOB1B2). Single-colony–derived clones were isolated and cultured with or without DI for 8 h. Lysates were prepared and analyzed with immunoblotting. Lysates from parental HeLa and cyclin B2 KO cells (^KOB2) were included as controls. Equal loading of lysates was confirmed by immunoblotting for actin. (C) Enhanced formation of cyclin B2–CDK1 and cyclin A–CDK1 complexes in the absence of cyclin B1. ^mAIDB1^KOB1 cells synchronized with a double thymidine block were cultured with or without DI and harvested at the indicated time points. Lysates were prepared and subjected to immunoprecipitation with an antibody against CDK1. Both total lysates and immunoprecipitates were analyzed with immunoblotting. (D) Indel analysis of cyclin B1 and cyclin B2. The endogenous cyclin B1 (CCNB1) and cyclin B2 (CCNB2) loci in ^mAIDB1^KOB1B2 cells were analyzed with sequencing. Sequencing traces of control (HeLa) and the edited samples were generated for indel analysis. The targeted sequence of the gRNA (solid black line), PAM sequence (dotted red line), and edited site (dotted black line) are indicated. Discordance, calculated by ICE, is shown for the edited (green) and control (orange) traces. The alignment window indicates the region of the traces with high Phred quality scores used for alignment. The inference window indicates the altered sequences around the edited site (dotted black line). Indel and corresponding prevalence were determined using ICE, with editing efficiencies of 90% for cyclin B1 and 95% for cyclin B2. (E) Efficiency of cyclin B1 silencing in ^mAIDB1^KOB1B2 cells. After treatment with DI for 6 h, lysates were prepared and analyzed with immunoblotting. Lysates from HeLa cells were included to serve as a reference for the expression level of endogenous cyclin B1. The signals corresponding to ^mAIDcyclin B1 were quantified using a standard curve based on serial dilutions of ^mAIDB1^KOB1B2 cell lysates (lanes 2–10), showing that <1% of ^mAIDcyclin B1 remained after DI treatment. (F) DI treatment does not affect the overall cell cycle distribution. HeLa cells were treated with DI for 24 h, pulsed with BrdU for 30 min, and analyzed using bivariate flow cytometry. Representative contour plots are shown (red: BrdU-positive; yellow: BrdU-negative S; blue: G₁; green: G₂/M). The positions of 2N and 4N DNA content are indicated. The percentage of cells at different cell cycle stage (excluding BrdU-negative S) was quantified. Mean and SEM from three independent experiments. (G) DI treatment does not affect cell cycle progression. Parental HeLa cells were treated with DI and analyzed using live-cell imaging for 48 h. The cumulative percentage of cells entering the first and second mitosis over time is shown. Source data are available for this figure: SourceData FS1.