Cas9 Stable Cell Line - HPAF-II

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer driven by mutations in genes such as RNF43, which plays a key role in Wnt signaling. The researchers used a genome-wide CRISPR-Cas9 screen in HPAF-II PDAC cells, which are sensitive to PORCN inhibition, to identify fitness genes required for cell proliferation. By generating HPAF-II-Cas9 cells expressing Streptococcus pyogenes Cas9, they performed a forward genetic screen using the TKO gRNA library, targeting over 17,000 human genes. This screen revealed several essential genes, including core components of the Wnt signaling pathway like WLS, CTNNB1, and PORCN, and highlighted the importance of the FZD5 receptor in RNF43-mutant PDAC cells.

Figure 1. The researchers conducted clonogenic proliferation assays in HPAF-II-Cas9 cells, transduced with lentivirus delivering specific gRNAs targeting FZD5. They confirmed gene editing through T7 endonuclease I cleavage assays and assessed cell viability in various PDAC cell lines. (Steinhart Z, et al., 2017)