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Multidrug resistance (MDR) has been extensively reported in colorectal cancer patients, which remains a major cause of chemotherapy failure. One of the critical mechanisms of MDR in colorectal cancer is the reduced intracellular drug level led by the upregulated expression of the ATP‐binding cassette (ABC) transporters, particularly, ABCB1/P‐gp. In this study, the CRISPR/Cas9 system was utilized to target ABCB1 in MDR colorectal cancer SW620/Ad300 cell line with ABCB1 overexpression. The results showed that stable knockout of ABCB1 gene by the CRISPR/Cas9 system was achieved in the MDR cancer cells. Reversal of MDR against ABCB1 chemotherapeutic drugs increased intracellular accumulation of [3H]‐paclitaxel accumulation, and decreased drug efflux activity was observed in MDR SW620/Ad300 cells after ABCB1 gene knockout. Further tests using the 3D multicellular tumor spheroid model suggested that deficiency in ABCB1 restrained tumor spheroid growth and restore sensitivity to paclitaxel in MDR tumor spheroids. Overall, the CRISPR/Cas9 system targeting the ABCB1 gene can be an effective approach to overcome ABCB1‐mediated MDR in colorectal cancer SW620/Ad300 cells., In this study, the CRISPR/Cas9 system was utilized to target ABCB1 in multidrug resistant (MDR) colorectal cancer SW620/Ad300 cell line with ABCB1 overexpression. Knockout of ABCB1 gene resulted in reversal of MDR against ABCB1 chemotherapeutic drugs, increased intracellular accumulation of [3H]‐paclitaxel accumulation, and decreased drug efflux activity in MDR colon cancer cells. In a 3D multicellular tumor spheroid model, deficiency in ABCB1 restrained tumor spheroid growth and restore sensitivity to paclitaxel in MDR tumor spheroids.

ATP‐binding cassette (ABC) transporters superfamily mediates multidrug resistance in cancer by extruding structurally distinct chemotherapeutic agents, causing failure in chemotherapy. Among the 49 ABC transporters, multidrug resistance protein 7 (MRP7 or ABCC10) is relatively new and has been identified as the efflux pump of multiple anticancer agents including Vinca alkaloids and taxanes. Herein, we construct and validate a homology model for human MRP7 based on the cryo‐EM structures of MRP1. Structure–function relationship of MRP7 was obtained from molecular dynamics simulations and docking studies and was in accordance with previous studies of ABC transporters. The motion patterns correlated with efflux mechanism were discussed. Additionally, predicted substrate‐ and modulator‐binding sites of MRP7 were described for the first time, which provided rational insights in understanding the drug binding and functional regulation in MRP7. Our findings will benefit the high‐throughput virtual screening and development of MRP7 modulators in the future., Homology modeling of MRP7 and predicted binding pocket characterization via docking analysis.