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. 2021 Apr 3;22(7):3741. doi: 10.3390/ijms22073741

Figure 5.

Figure 5

Editing of PSMB6-YFP is improved in HeLa TAFts cells. HeLa TAFts cells were transfected with Cas9/sgRNA encoding plasmids and with donor DNA. Two days after transfection, cells were placed at restrictive or permissive temperatures. (A) PCR analysis of HeLa PSMB6-YFP cells. Genomic DNA from naïve HeLa cells and co-edited non-selected or selected pools were subjected to PCR using the primers indicated. The “1” forward and “2” reverse primers generate a 600 bp fragment using naïve genomic template and a 1500 bp fragment with the YFP insertion. Using the YFP reverse primer (“3”) and “1” forward primer, the expected fragment is 1050 bp. Using “1” forward and “4” reverse primers, a 300 bp fragment is generated and used for normalization. (B) FACS analysis of HeLa PSMB6-YFP co-edited cells. Cells were analyzed by FACS with 30,000 cells per point, n = 3 *** p < 0.0001. (C) PSMB6-YFP is enriched in HeLa co-edited cells. Pools of selected (39.5 °C) and unselected (37 °C) cells transfected with the reagents indicated were analyzed by SDS–PAGE and immunoblotting with the indicated antibodies.