Double-Stranded Biotinylated Donor Enhances Homology-Directed Repair in Combination with Cas9 Monoavidin in Mammalian Cells

Philip J R Roche; Heidi Gytz; Faiz Hussain; Christopher J F Cameron; Denis Paquette; Mathieu Blanchette; Josée Dostie; Bhushan Nagar; Uri David Akavia

doi:10.1089/crispr.2018.0045

Double-Stranded Biotinylated Donor Enhances Homology-Directed Repair in Combination with Cas9 Monoavidin in Mammalian Cells

CRISPR J. 2018 Dec:1:414-430. doi: 10.1089/crispr.2018.0045.

Authors

Philip J R Roche¹, Heidi Gytz², Faiz Hussain¹, Christopher J F Cameron^{1

3

4}, Denis Paquette¹, Mathieu Blanchette⁴, Josée Dostie^{1

3}, Bhushan Nagar^{1

5}, Uri David Akavia^{1

3}

Affiliations

¹ 1 Department of Biochemistry, McGill University, Montreal, Canada.
² 2 Department of Molecular Biology and Genetics, Aarhus University , Aarhus, Denmark.
³ 3 Rosalind and Morris Goodman Cancer Research Centre, McGill University , Montreal, Canada.
⁴ 4 School of Computer Science and Centre for Bioinformatics, McGill University , Montreal, Canada.
⁵ 5 Groupe de Recherche Axé sur la Structure des Protéines, McGill University , Montreal, Canada .

PMID: 31021244
DOI: 10.1089/crispr.2018.0045

Abstract

Homology-directed repair (HDR) induced by site specific DNA double-strand breaks 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 a Cas9 protein fused to a monoavidin domain to bind biotinylated donor DNA. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9-donor DNA complexes into cells. Improved HDR percentages of up to 90% in three loci tested (CXCR4, EMX1, and TLR) in standard HEK293T cells were observed. Our results suggest that donor DNA biotinylation and Cas9-donor conjugation in addition to delivery influence HDR efficiency.