Cell‐Penetrating Peptide‐Based Triple Nanocomplex Enables Efficient Nuclear Gene Delivery in <i>Chlamydomonas reinhardtii</i>

Microalgae are a promising solution for mitigating climate change due to their ability to capture greenhouse gases and produce renewable materials. However, their effective application is often hindered by barriers that necessitate advances in genetic engineering to improve photosynthesis and productivity. One major obstacle is the microalgal cell wall, which complicates the delivery of genetic material into these organisms. To address these challenges, we developed a novel triple nanocomplex system integrating cell-penetrating peptides (CPPs), nuclear localization signal (NLS) peptides, and plasmid DNA. This system allows simple preparation while achieving efficient nuclear translocation of plasmid DNA. We evaluated two CPPs, pVEC-ORI and pVEC-R6A, for their efficacy in facilitating intracellular transfer of DNA into wild-type Chlamydomonas reinhardtii cells. Notably, pVEC-R6A demonstrated a 6.88-fold increase in efficiency compared to pVEC-ORI, despite the presence of thick cell walls. The optimal CPP:DNA ratio for stable nanocomplex formation was determined to be 5:1 for pVEC-ORI and 10:1 for pVEC-R6A. By incorporating the simian virus 40 (SV40) NLS into CPP/DNA nanocomplexes, we successfully directed the localization of plasmid DNA into the nucleus. Our findings indicate that this simple and efficient DNA delivery system has significant potential as a tool to advance microalgal synthetic biology.