PT  - JOURNAL ARTICLE
AU  - Philip J.R. Roche
AU  - Heidi Gytz
AU  - Faiz Hussain
AU  - Christopher J.F. Cameron
AU  - Denis Paquette
AU  - Mathieu Blanchette
AU  - Josée Dostie
AU  - Bhushan Nagar
AU  - Uri David Akavia
TI  - Efficient Homology Directed Repair by Cas9:Donor Localization and Cationic Polymeric Transfection in Mammalian Cells
AID  - 10.1101/248179
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 248179
4099  - http://biorxiv.org/content/early/2018/01/19/248179.short
4100  - http://biorxiv.org/content/early/2018/01/19/248179.full
AB  - 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 repair. In order to obtain high HDR frequency in mammalian cells, we delivered donor DNA to a DSB site by engineering a Cas9 protein fused to a high-affinity monoavidin domain to accept biotinylated DNA donors. In addition, we used the cationic polymer, polyethylenimine, to efficiently deliver our Cas9-DNA donor complex into the nucleus, thereby avoiding drawbacks such as cytotoxicity and limited in vivo translation associated with the more commonly used nucleofection technique. Combining these strategies led to an improvement in HDR rates of up to 90% on several test loci (CXCR4, EMX1, TLR). Our approach offers a cost effective, simple and broadly applicable editing method, thereby expanding the CRISPR/Cas9 genome editing toolbox.