CRISPR/Cas9 screen to identify genes involved in uptake of cell-penetrating peptides (Raji)

Cell-permeable peptides (CPPs) allow intracellular delivery of cargo molecules that are hooked to them. CPPs provide an efficient methodology to transfer bioactive molecules in cells, in particular in conditions when transcription or translation of cargo-encoding sequences is not desirable or achievable. The mechanisms allowing CPPs to enter cells are ill-defined and controversial. No genes have been described that regulate the intracellular delivery of CPPs. Using a CRISPR/Cas9-based screening, we discovered that the KCNQ5, KCNN4, and KCNK5 potassium channels are required for direct cellular translocation of TAT-RasGAP317-326 anti-cancer compound in various cell lines by setting the appropriate transmembrane potential of the cells. These potassium channels also affected the cellular uptake of other TAT-bound cargos.

细胞穿透肽（cell-permeable peptides, CPPs）能够实现与其偶联的载荷分子的胞内递送。细胞穿透肽为生物活性分子的细胞内递送提供了高效策略，尤其适用于无法或不宜对载荷编码序列进行转录或翻译的场景。细胞穿透肽进入细胞的具体机制迄今尚未明确，且存在诸多争议。目前尚无调控细胞穿透肽胞内递送的相关基因被报道。本研究通过基于CRISPR/Cas9的筛选实验发现，KCNQ5、KCNN4与KCNK5三种钾离子通道，可通过调控细胞的适宜跨膜电位，介导TAT-RasGAP317-326抗癌化合物在多种细胞系中的直接细胞转位过程。上述钾离子通道同样可影响其他与TAT偶联的载荷分子的细胞摄取效率。