OFF 3 FECO 1
To assess whether the improved TESO construct, Cas9-FECO fusion, induces additional off-target effects, we conducted a second experiment. WT Cas9 and Cas9-FECO were electroporated into K562 cells as mRNA, and the cells were recovered for five days before analysis. Additionally, we co-delivered a circular single-stranded DNA donor containing a GFP sequence targeting Rab11A to evaluate potential HDR activity. We selected the four most likely off-target sites based on computational predictions and designed NGS amplicons flanking each site. Following sequencing, we analyzed the off-target mutation frequency and patterns. The results indicated that both WT Cas9 and Cas9-FECO induced only minimal off-target effects. The sequences of the four off-target sites and the results of this experiment are presented in Supplementary Figure 4 of the manuscript Engineering Tripartite Gene Editing Machinery for Highly Efficient Non-Viral Targeted Genome Integration.