Abstract Background and Purpose Due to the complexity of traditional Chinese medicine based on complex mixtures of natural products and their multi-target mechanism of action, the discovery and validation of relevant targets have always been challenging. In previous studies, using transcriptomic methods and Compound Kushen Injection (CKI) as a model drug, we identified multiple pathways and target genes through which CKI exerts its pharmacological effects. Therefore, we wished to verify these targets by perturbing those genetic pathways. Experimental Approach In this study, we selected eight key genes from four candidate pathways and used CRISPR/CAS technology to knock out these genes in four cell lines, validating their role in CKI activity. Key Results Firstly, we found that although the sensitivity of different cell lines to gene knockout varied, overall, it led to a reduction in various cellular activities. After the addition of CKI, we observed that, except for the minor impact of CDKN1A gene knockout on the effect of CKI, knocking out the other genes significantly affected the pharmacological efficacy of CKI in different assays. Among them, knockout of MYD88 and NFkB genes enhanced the efficacy of CKI. At the same time, we found that the genes IL24 and CYP1B1 play a crucial role in CKI inhibition of tumour cell migration, and the CYP1A1 gene is critical for the cell cycle arrest induced by CKI. Conclusion and Implications These find ing s not only validate the results of our previous transcriptomic analysis but also further demonstrate the complexity of pharmacological mechanisms of multi-target synergistic action of natural product mixtures. What is already known CKI demonstrates antitumor effects in both clinical and pharmacological research. Transcriptomic analysis shows CKI can perturb the expression of numerous genes in pathways related to cancer. What does this study add The knockout of most selected genes whose expression is altered by CKI can significantly affect the pharmacological effects of CKI. IL24 and CYP1B1genes are essential to CKI’s inhibition of cancer cell migration and CYP1A1 is important for CKI’s G2M cell cycle arrest effect. What is the clinical significance The efficacy of CKI is demonstrated to arise from the synergistic action of multiple pathways and targets.

Validating the Target Functions and Synergistic Multi-Target, Multi-Pathway Action Mode of Compound Kushen Injection Using CRISPR/CAS