Analysis of the effects of human wild-type TREX1 and 3' frameshift mutant TREX1 (RVCL TREX1) expression on CRISPR/Cas9-induced DSB repair by short amplicon sequencing.
We used 293 cells stably expressing one copy of wild-type TREX1 or 3' frameshift mutant TREX1 (RVCL TREX1) in a doxycycline (Dox)-inducible manner using the Flp-in expression system. Double-strand breaks (DSB) were induced by CRISPR/Cas9 in each cell line expressing exogenous TREX1, and the deletion status at the cut site after DSB repair was analyzed by short amplicon sequencing. Sequence analysis was performed by short PCR amplicon sequencing using illumina MiSeq (2Ã?300 bp). The target gene was human ACTB, which was amplified using a primer flanking the cut site. Sequencing analysis was performed on an amplicon of approximately 500 bp. The sample groups consisted of four groups: Dox-untreated wild-type TREX1-expressing cells and RVCL-expressing cells as controls, and each cell line sample that was Dox-treated and expressed exogenous TREX1. Three biological replicates were obtained for each group and the total sequence data consisted of 12 samples.