The same zPEIs also provided 16-fold greater efficiency of CRISPR/Cas9 gene knock-in compared to unmodified PEI in the presence of serum. Such polymer modifications highlight the need for a more fundamental understanding of the complex correlations between polycation-DNA interactions and the resulting physicochemistry of polyplexes and transgene expression observed in cells. A better understanding of the critical barriers to nonviral gene delivery is required to improve efficacy in vivo and ultimately move nonviral vectors into clinical applications.