Generation of a Double Gene Knockout Cell Line Model in Hard-to-Edit Jurkat Cell Lines

Figure 1. A sequential targeting strategy was used to disrupt two genes of interest in Jurkat (suspension) cells. First, exon 2 of gene #1 was disrupted using 2 gRNAs (g1.1 and g1.2) and single cell clones were identified. The identified gene #1 knockout (KO) clones were re-targeted with two different gRNAs (g2.1 and g2.2) for gene #2. Single cell clones were isolated and confirmed to have double KO deletion by PCR and sequencing.

Figure 2. Representative clone from gene #1 KO has a 215 bp insertion that causes a frame shift mutation. Three out of 40 clones were identified as gene #1 KO and subsequently used for KO of gene #2.

Figure 3. After the second targeting experiment, one clone was identified as a gene #2 KO. This clone contained a 115 bp insertion at the targeted locus that caused a frame shift mutation and disruption of gene expression.