Knock-In iPS Cell Line Generation Using End-Modified Linear DNA Donors

Aidan M. Simpson1, Christopher B. Rohde2, Mitchell R. Kopacz3, Jasmine K. Harris2, Franklin Kostas2, I. Caglar Tanrikulu3, Matthew Angel2

1Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 2Factor Bioscience, Inc., Cambridge, MA, 3Novellus, Inc., Cambridge, MA

Mol Ther, Vol 29, No 4S1, 2021

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.

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