CRISPR/Cas9 generation of HADHA KO PANC-1 cells to study the role of HADHA in pancreatic cancer stemness

This study employs the CRISPR/Cas9 technique to generate HADHA knockout (KO) PANC-1 cells to investigate the role of the HADHA gene in the stemness of pancreatic cancer stem cells (PCSCs). Utilizing Lipofectamine 3000 for transfection, a specific clone (C8E12) was identified and validated via Western Blot and Sanger sequencing, revealing significant biallelic deletions. The impact of HADHA deficiency was evaluated by culturing cells in a stem-selective medium to induce tumoursphere formation. The results indicate that HADHA knockout does not alter the fundamental stemness of the PANC-1 cell line, as the KO clone retained the ability to grow in suspension and remain undifferentiated. However, HADHA-KO PCSCs formed more compact and denser tumourspheres compared to wild-type cells. These morphological differences suggest that while HADHA is not essential for maintaining stemness, its absence significantly affects the structural characteristics of PCSCs. Further investigation is required to determine the impact of HADHA knockout on PCSC proliferation, chemoresistance, and migration to fully understand its potential as a therapeutic target in pancreatic cancer.