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Simple Summary: Knocking down genes by shRNAs in CAR T cells offers the potential of expanding the therapy's efficacy beyond its initial success. Since shRNAs are in the CAR construct, only the CAR specifically will be affected by the knockdown. Due to that intrinsic nature, we show that these knockdowns can make CAR T cells resistant to HIV and chemo-agents. Like other gene editing tools, such as CRISPR, shRNAs need to be optimized. In this article, we elucidate four common design optimizations necessary to construct a fully functional shRNA-containing CAR. Short hairpin RNAs (shRNAs) have emerged as a powerful tool for gene knockdown in various cellular systems, including chimeric antigen receptor (CAR) T cells. However, the elements of shRNAs that are crucial for their efficacy in developing shRNA-containing CAR T cells remain unclear. In this study, we evaluated the impact of different shRNA elements, including promoter strength, orientation, multiple shRNAs, self-targeting, and sense and antisense sequence composition on the knockdown efficiency of the target gene in CAR T cells. Our findings highlight the importance of considering multiple shRNAs and their orientation to achieve effective knockdown. Moreover, we demonstrate that using a strong promoter and avoiding self-targeting can enhance CAR T cell functionality. These results provide a framework for the rational design of CAR T cells with shRNA-mediated knockdown capabilities, which could improve the therapeutic efficacy of CAR T cell-based immunotherapy. [ABSTRACT FROM AUTHOR]