Novel regulation on the mitotic checkpoint revealed by knocking out CDC20

To ensure accurate segregation of genetic materials during cell division, eukaryotic cells employ a highly conserved signaling pathway called the spindle assembly checkpoint (SAC) to monitor the generation of a stable attachment between microtubules and kinetochores. Unattached kinetochores activate the checkpoint by recruiting multiple checkpoint proteins, which further catalyze the formation of the mitotic checkpoint complex (MCC). Composed of BubR1, Bub3, Mad2, and Cdc20, MCC directly binds to and inhibits the anaphasepromoting complex/cyclosome (APC/C), the E3 ligase responsible for the ubiquitination of key mitotic regulators like cyclin B1 and securin. High Cdk1/cyclin B1 kinase activity retains the cells in mitosis; thus, the cells are given more time to generate a stable kinetochore–microtubule attachment. Once the stable attachment is generated, the checkpoint is silenced from the kinetochores and the produced MCC is disassembled via multiple mechanisms. Cyclin B1 and securin gets degraded, and cells enter anaphase with coordinated chromatid segregation (Musacchio, 2015; Lara-Gonzalez et al., 2021; McAinsh and Kops, 2023). However, even in the persistence of an active checkpoint, cells could still exit mitosis and enter interphase without proper chromosome segregation and cytokinesis. This unusual mitotic exit is called mitotic slippage and yields tetraploid multinucleated cells which could either die after the slippage or arrest in the G1 phase, or continue the cell cycle. Mitotic slippage could be caused by the gradual degradation of cyclin B1 by the leakage activity of APC/C or by the weakening of SAC (Topham and Taylor, 2013). As a key mitotic regulator, Cdc20 plays opposite roles in mitosis as one molecule binds and activates APC/C to promote anaphase initiation and another molecule forms MCC to inhibit APC/C and delay anaphase initiation (Izawa and Pines, 2015; Alfieri et al., 2016). Although Cdc20 has been extensively studied, its functional investigation within cells is limited by the fact that mammalian cells only need a minimal amount of Cdc20 for cell division and that RNAi against Cdc20 could not efficiently deplete the protein to induce a penetrant phenotype (Wolthuis et al., 2008; Baumgarten et al., 2009). Meanwhile, complete inactivation of CDC20 causes metaphase arrest, followed by apoptosis; thus, a genuine knockout cell line could not be generated (Li et al., 2007). CRISPR/Cas9-mediated gene knockout has been widely applied in gene function analysis. By introducing DNA fragment insertion or deletion (indels), the targeted gene is disrupted as the reading frame is shifted and a premature termination codon (PTC) occurs (Doudna and Charpentier, 2014). Until now, numerous knockout cell lines or organisms have been generated, which significantly promoted the gene function investigation. However, mounting pieces of evidence show that disrupting the reading frame may not fully silence the targeted genes. Truncated proteins could be produced via two distinct mechanisms to partially or fully compensate the putative loss of function. Translation OPEN ACCESS