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

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

Open Access

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

Author(s) -

Philip J. R. Roche,

Heidi Gytz,

Faiz Hussain,

Christopher JF Cameron,

Denis Paquette,

Mathieu Blanchette,

Josée Dostie,

Bhushan Nagar,

Uri David Akavia

Publication year - 2018

Publication title -

the crispr journal

Language(s) - English

Resource type - Journals

eISSN - 2573-1602

pISSN - 2573-1599

DOI - 10.1089/crispr.2018.0045

Subject(s) - biotinylation , cas9 , homology directed repair , dna , hek 293 cells , crispr , polyethylenimine , genome editing , microbiology and biotechnology , chemistry , dna repair , transfection , biology , gene , nucleotide excision repair , biochemistry

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.

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