Updated project metadata. Accurate chromosome segregation is coordinated by the spindle assembly checkpoint (SAC) through its effector the mitotic checkpoint complex (MCC), to inhibit the anaphase-promoting complex or cyclosome (APC/C). Cdc20 is an essential mitotic regulator since it promotes mitotic exit through activating the APC/C and monitors kinetochore-microtubule attachment through activating the SAC. The proper functioning of Cdc20 requires multiple interactions with APC/C and MCC subunits. To functionally assess each of these interactions within cells requires efficient depletion of endogenous Cdc20, which is highly difficult to achieve by RNAi. Here we generated Cdc20 RNAi sensitive cell lines by CRISPR/Cas9 which display a penetrant metaphase arrest phenotype by a single RNAi treatment. In this null background, we accurately measured the contribution of each known motif of Cdc20 on APC/C and SAC activation. The CRY box, a previously identified degron was found to be critical for the SAC by promoting theMCC formation and stabilizing the interaction between the MCC and APC/C. These data reveal additionalregulatory components within the SAC and establish a novel method to interrogate Cdc20 function.