Knock-In iPS Cell Line Generation Using End-Modified Linear DNA Donors
To produce a targeted knock-in, a gene-editing endonuclease is used to create a double strand break (DSB) at a target site in the genome, and a plasmid donor containing a transgene as well as homology arms is inserted at the target site. However, rates of on-target integration using plasmid donors are very low, especially in primary cells and induced pluripotent stem cells (iPSCs). We hypothesized that the use of end-modified linear DNA donors could result in higher insertion rates and increased target specificity when compared with traditional plasmid donors. We synthesized end-modified linear donors using PCR with standard primers, 5’-biotinylated primers, and primers containing a 5′ polyethylene glycol (PEG) linker connected to a random 21-nucleotide single-stranded DNA sequence. We hypothesized that these donors could exhibit lower cytotoxicity, increased persistence in cells, and improved rates of on-target integration. To examine these characteristics, we compared the three donors in knock-in experiments using primary human fibroblasts and iPSCs. End-modified linear donors encoding green fluorescent protein (GFP) and a puromycin resistance gene were synthesized and electroporated into fibroblasts and iPSCs together with mRNA encoding NoveSlice gene-editing proteins targeting a sequence within the AAVS1 locus. We optimized the electroporation parameters and cell culture conditions for simultaneous delivery of gene-editing mRNA and donor DNA to iPSCs. Using GFP expression levels as a marker, the standard PCR donor resulted in the highest insertion rate, followed by the 5′ ssPEG donor and the biotinylated donor. All three donors resulted in higher integration rates than a plasmid containing the same sequence. Transfected iPSCs formed colonies of cells with uniform GFP expression that were isolated and propagated as stable knock-in lines. We show that end-modified linear donors integrate at higher rates than plasmid donors. Use of these donors may therefore represent a preferred approach for the generation of knock-in iPS cell lines.