CRISPR-mediated MTTP Knockout in Huh-7 Cells

To knockout the MTTP gene by plasmid-based CRISPR system, plasmids expressing gRNA and CRISPR/Cas9 KO plasmids were co-transfected with MTP-HDR plasmids (1:1 ratio) into Huh-7 cells by chemical method using EndoFectin Max transfection reagent (Genecopoeia, #EF013) as described earlier (8 (link), 10 (link), 27 (link)). Huh-7 cells were seeded in T75 flasks and allowed to grow until they reached 80%–90% confluence. Cells were trypsinized, washed with OptiMem media, and 10⁶ cells were resuspended in 5 ml of OptiMem media. The MTP CRISPR/Cas9 KO (5 μg), MTP-HDR plasmids (5 μg), and EndoFectin (20 μl) were mixed in 0.5 ml of OptiMem media. The mixture was incubated at room temperature for 10 min and added to 90 mm cell culture dishes. Cells were then added to the plate. After 12 h incubation in CO₂ incubator at 37°C, the OptiMem media were removed, and DMEM media was added. Similarly, the transfection of pcDNA3 or pcDNA3-hMTP-FLAG (9 μg of each) into Huh7 MTP KO cells was carried out using EndoFectin Max transfection reagent (Genecopoeia, #EF013).