Homology Directed Repair by Cas9:Donor Co-localization in Mammalian Cells
Philip JR Roche,
Christopher J.F. Cameron,
Uri David Akavia
Posted 16 Jan 2018
bioRxiv DOI: 10.1101/248179 (published DOI: 10.1089/crispr.2018.0045)
Posted 16 Jan 2018
Homology directed repair (HDR) induced by site specific DNA double strand breaks (DSB) with CRISPR/Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered Cas9 protein fused to a high-affinity monoavidin domain to deliver biotinylated donor DNA to a DSB site. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9 RNP-donor DNA complex into the cell. Combining these strategies improved HDR percentages of up to 90% in three tested loci (CXCR4, EMX1, and TLR) in standard HEK293 cells. Our approach offers a cost effective, simple and broadly applicable gene editing method, thereby expanding the CRISPR/Cas9 genome editing toolbox.
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