In non-viral gene delivery, DNA entering the cytoplasm is rapidly bound by barrier-to-autointegration factor (BAF) and surrounded by nuclear envelope-like membranes. This process is thought to mitigate the translocation of the DNA from the cytoplasm through the nuclear pores limiting the transfection of interphase cells. To prevent BAF clustering, we developed the protein-based gene delivery system TFAMoplex that uses the human mitochondrial transcription factor A (TFAM) which forms nanoparticles with DNA. In previous studies, we covalently fused the endogenous BAF inactivating enzyme vaccinia-related kinase 1 (VRK1) to TFAM, which, however, did not decrease BAF-mediated clustering of the transfected DNA. Here, we describe a modified TFAMoplex where VRK1 is linked to TFAM via a SpyTag/SpyCatcher linker system (SpyTFAMoplex). With this approach, quantitative image analysis, performed with transfected enhanced green fluorescent protein (EGFP)-BAF overexpressing HeLa cells, showed that cytoplasmic BAF clustering can be counteracted. A SpyTFAMoplex version comprising a kinase-inactive VRK1 mutant (dSpyTFAMoplex) exhibited twice the number of clusters per cell. The decrease in BAF clusters achieved with the active VRK1 system was accompanied by lower transfection efficiency in HeLa cells with significantly less (−72 %) mean fluorescence intensity of reporter gene expression compared to dSpyTFAMoplex. These data suggest that VRK1 in the SpyTFAMoplex interferes with BAF's DNA-binding ability, potentially impairing the mitotic nuclear entry pathway of exogenous DNA. While our study highlights the potential of co-delivering enzymes with DNA, further vector engineering is required to directly guide cytosolic DNA into the nucleus of interphase cells.