ENAapplication/xmlftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR328/003/SRR32811403/SRR32811403_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR328/005/SRR32811405/SRR32811405_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR328/099/SRR32811399/SRR32811399_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR328/005/SRR32811405/SRR32811405_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR328/001/SRR32811401/SRR32811401_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR328/002/SRR32811402/SRR32811402_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR328/003/SRR32811403/SRR32811403_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR328/098/SRR32811398/SRR32811398_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR328/000/SRR32811400/SRR32811400_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR328/098/SRR32811398/SRR32811398_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR328/002/SRR32811402/SRR32811402_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR328/000/SRR32811400/SRR32811400_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR328/004/SRR32811404/SRR32811404_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR328/004/SRR32811404/SRR32811404_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR328/001/SRR32811401/SRR32811401_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR328/099/SRR32811399/SRR32811399_1.fastq.gzprimary200OKGenomicsnortheastern universityhttps://www.ebi.ac.uk/ena/browser/view/PRJNA1240329We 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.ENAfalseEngineering Tripartite Gene Editing Machinery for Highly Efficient Non-Viral Targeted Genome Integration2025-03-292025-03-29PRJNA1240329