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 further investigate their impact on Cas9 cutting efficiency and indel patterns, we conducted a CRISPR-Cas9 experiment using a normalized amount of plasmid DNA and electroporation in K562 cells. Cas9 was delivered as an all-in-one plasmid containing both Cas9 and the Rab11A gRNA. Following electroporation, cells were recovered for five days before analysis. We targeted the Rab11A locus, testing various Cas9 fusion proteins alongside WT Cas9 as a positive control and a mock condition as a negative control. After cell lysis, the Rab11A site was sequenced using Illumina NGS with amplicons flanking the cut site. The raw sequencing data was processed using the CRISPRESSO2 web server for indel percentage and pattern analysis. The results showed that the short motif fusion exhibited similar cutting efficiency to WT Cas9, whereas fusions with larger proteins resulted in reduced cutting efficiency. The results of this experiment are presented in Supplementary Figure 1 of the manuscript Engineering Tripartite Gene Editing Machinery for Highly Efficient Non-Viral Targeted Genome Integration.