Analysis of the effects of human wild-type TREX1 and 3' frameshift mutant TREX1 (RVCL TREX1) expression on CRISPR/Cas9-induced DSB repair.
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 long-read sequencing. Sequence analysis was performed by acquiring HiFi reads using the PacBio Revio/Sequel IIe. 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 3800 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.