Loss of KNTC1 Triggers a 53BP1 and USP28 Dependent Cell Cycle Arrest Without Prolonged Mitotic Delays

While the spindle assembly checkpoint (SAC) ensures mitotic fidelity by delaying mitotic progression, a SAC-independent surveillance program is known to monitor the duration – without affecting the progression – of mitosis. This pathway, mediated by 53BP1, USP28, and p53, induces post-mitotic arrest in daughter cells following mitoses that exceed a defined temporal threshold.

To uncover additional inputs into this pathway, we performed genome-wide CRISPR-Cas9 screens in wild-type, p53-/-, and USP28-/- human cells. Among genes selectively required in wild-type cells, we identified two components of the RZZ kinetochore complex—KNTC1 (ROD) and ZWILCH—as high-confidence hits. Remarkably, depleting KNTC1, which impaired SAC maintenance, triggered a robust 53BP1, USP28, and p53-dependent arrest without the characteristic mitotic delay.