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

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DOI

  • Philip J.R. Roche, Department of Biochemistry, McGill University, Montreal, Canada
  • Heidi Gytz
  • Faiz Hussain, Department of Biochemistry, McGill University, Montreal
  • ,
  • Christopher J.F. Cameron, Department of Biochemistry, McGill University, Montreal, Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal, School of Computer Science and Centre for Bioinformatics, McGill University, Montreal
  • ,
  • Denis Paquette, Department of Biochemistry, McGill University, Montreal
  • ,
  • Mathieu Blanchette, School of Computer Science and Centre for Bioinformatics, McGill University, Montreal
  • ,
  • Josée Dostie, Department of Biochemistry, McGill University, Montreal, Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal
  • ,
  • Bhushan Nagar, Department of Biochemistry, McGill University, Montreal, Department of Biochemistry, Groupe de Recherche Axé sur la Structure des Protéines, McGill University, Montreal, Canada.
  • ,
  • Uri David Akavia, Department of Biochemistry, McGill University, Montreal, Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal
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.
Original languageEnglish
JournalThe CRISPR Journal
Volume1
Issue6
Pages (from-to)414-430
Number of pages17
ISSN2573-1599
DOIs
Publication statusPublished - 20 Dec 2018

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