Figure 3.

SiC Reduces Off-Target Editing

(A) HEK293T cells stably expressing vector SiC-V2 with Cas9-G7 were transduced with SiC-V1 carrying previously established promiscuous sgRNA targeting either EMX1 or VEGFA. Both guides have well-established on-target (T) and off-target (OT) editing sites. 48 h post transduction, the cells were divided into two groups with one group receiving 1 μg/mL Dox for another 13 days. Genomic DNA was prepared from these four types of cells at the end point and also control untransduced HEK293T-Cas9G7cells cultured with Dox. (B) 6-FAM labeled PCR products generated for EMX1 and VEGFA, on-target and off-target sites, on day 15 were resolved using capillary electrophoresis. Vertical red line indicates unedited PCR fragment from control cells (top row). Numbers in individual panels indicate percent editing based on electrophoresis area-under-the-curve calculations. Off-target editing is reduced upon Dox addition (B, middle versus bottom row). Data are representative of duplicate runs.