4.4.3. Lymphocyte Transfection in a 12-Well Plate Using Lipofectamine CRISPRMAX Reagent (cat. no. CMAX00008, Thermofischer, Waltham, MA, USA)

At the transfection day, 1.5 mL sterile Eppendorf tube was filled with 50 μL of Opti-MEM medium (cat. no. 31985062), then 1.25 µg GeneArt Platinum Cas9 nuclease and 0.25 µg gRNA were added followed by mixing using vortex, after that, 2.5 μL Cas9 Plus reagent was added to the mixture that contained Cas9 protein and gRNA, mixed well, and incubated at 25 °C for 5 min to form RNPs. Meantime, 3 µL of Lipofectamine CRISPRMAX was mixed with 50 µL Opti-MEM and incubated at 25 °C for 5 min before being added to the RNP solution. The mixture was incubated at 25 °C for 15 min, then added to cells that were plated onto 12-well plates at a density of 8.5 × 10⁵ cells/well in 1 mL growth medium. At 72 h post-transfection, lymphocytes were harvested for cell count and viability using the trypan blue exclusion method, and gene expression was analyzed before and after editing [36]. The genomic cleavage efficiency was measured by the GeneArt Genomic Cleavage Detection kit (cat. no. {"type":"entrez-protein","attrs":{"text":"A24372","term_id":"80014","term_text":"pir||A24372"}}A24372, Thermofischer, Waltham, MA, USA).