Quantification of gene-editing efficiency by Sanger sequencing

Genomic DNA of H1299-EGFP or primary human T cells was extracted using the QIAamp DNA Blood Mini Kit (QIAGEN, Chatsworth, CA, USA), according to the manufacturer’s instructions. Next, the DNA target sites of interest were amplified by PCR using KAPA HiFi HotStart ReadyMix (Roche Diagnostics Belgium, Diegem, Belgium). Amplified PCR products were purified by the QIAquick PCR purification kit (QIAGEN, Chatsworth, CA, USA), following the manufacturer’s protocol. Next, PCR amplicons were sequenced by the GATC lightrun service (Eurofins Genomics, Ebersberg, Germany), after which the sequencing data were analyzed using TIDE (http://shinyapps.datacurators.nl/tide/).55