Engineering Tripartite Gene Editing Machinery for Highly Efficient Non-Viral Targeted Genome Integration

We developed Cas9 fusion proteins containing single-stranded DNA-binding proteins and a 20-amino acid motif with single-stranded DNA-binding function. These fusion proteins demonstrated higher efficiency in Cas9-directed homology-directed repair (HDR) using circular single-stranded DNA as a donor template.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. 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.