P7 Enhanced CRISPR/Cas9-mediated precise genome editing

P7 Enhanced CRISPR/Cas9-mediated precise genome editing

Jason Potter1, Xiquan Liang1, Shantanu Kumar1, Namritha Ravinder1, Anja Martinez2, Julie Wood3 and Jonathan D Chesnut1

1Synthetic Biology R&D Thermo Fisher Scientific, Carlsbad, USA
2Synthetic Biology, Thermo Fisher Scientific, Stockholm, SWE
3Synthetic Biology, Thermo Fisher Scientific, London, GBR

While CRISPR-based gene knockout in mammalian cells has proven to be very efficient, precise insertion of genetic elements via the cellular homology directed repair (HDR) pathway remains a rate-limiting step to seamless genome editing. We now present up to 56% targeted integration efficiency of up to a six-nucleotide insertion in HEK293 cells. In iPSCs we achieved precise genome editing rates of up to 45% by co-delivering the Cas9 RNP and donor DNA. Also, the use of a dsDNA oligonucleotide with 3’ overhangs allowed integration of a longer tag at rates of up to 50%. We propose a model favoring donor DNAs with the change as close to the cleavage site as possible. For small changes, asymmetric single-stranded donors seem to be favored. For larger insertions (e.g. FLAG tag), a dsDNA donor with protruding 3’ homology arms of 30 bases is favored. Modifying the donor DNA ends with phosphorothioate further improves the editing efficiency.