shortly ampicated DNA of mus musculus: crRNP-transfected CD4 T cells
crRNA and tracrRNA are denatured in a PCR machine at 95C for 5 minutes, followed by annealing of the crRNA-tracrRNA duplex through a 1-hour incubation at room temperature. Cas9 is then added to form the crRNP complex. CD4 T cells, isolated from the spleen of a mouse, are cultured for approximately 42 hours on plates coated with anti-CD3 and anti-CD28. Afterward, the cells are harvested, and 3 x 10^6 cells are electroporated with the crRNP complex. The cells are then cultured for another 2 days before being sorted using a FACS sorter. Finally, gDNA is extracted from the cells, PCR is performed, and sequencing analysis is conducted.