Multiplexed CRISPR/Cas9 genome editing increases the efficacy of homologous-dependent repair of donor sequences in mammalian cells

Ezio T. Fok; Clement B. Penny; Musa M. Mhlanga; Marc S. Weinberg

doi:10.17159/sajs.2015/20150002

Authors

Ezio T. Fok Medical Oncology Research Unit, Department of Internal Medicine, School of Clinical Medicine, University of the Witwatersrand, Johannesburg, South Africa
Clement B. Penny Medical Oncology Research Unit, Department of Internal Medicine, School of Clinical Medicine, University of the Witwatersrand, Johannesburg, South Africa
Musa M. Mhlanga 1. Gene Expression and Biophysics Group, Synthetic Biology–Emerging Research Area, Council for Scientific and Industrial Research, Pretoria, South Africa 2. Unit of Biophysics and Gene Expression, Institute of Molecular Medicine, Faculty of Medicine, University of Lisbon, Lisbon, Portuga
Marc S. Weinberg 1. Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, School of Pathology, University of the Witwatersrand, Johannesburg, South Africa 2. HIV Pathogenesis Research Unit, Department of Molecular Medicine and Haematology, School of Pathology, University of the Witwatersrand, Johannesburg, South Africa 3. Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA

DOI:

https://doi.org/10.17159/sajs.2015/20150002

Keywords:

genome repair, genome targeting, sgRNA, HDR, T7 endonuclease I

Abstract

Efficient and robust genome editing tools and strategies allow for specific and exact genetic changes to be captured in model systems, thereby accelerating both forward and reverse genetics studies. The development of CRISPR/Cas9 as a facile designer nuclease toolset has allowed for defined genetic modifications to be efficiently made through homology-directed repair of targeted DNA double-stranded breaks (DSBs) using exogenous repair templates. However, traditional single DSB strategies are still relatively inefficient as the short gene conversion tracts of mammalian cell systems limit the extent of achievable gene alteration from the DSB site. In order to improve on the inefficiency, we devised a dual cut strategy, which relies on reconstituting entire deleted gene fragments to precisely modify extensive gene regions of interest. Using the CRISPR/Cas9 system, we were able to introduce targeted deletions and repair of the endogenous KRAS gene locus in cell culture. The use of two simultaneous DSBs can be employed for efficient application of homology-directed repair with a large dsDNA donor sequence, thereby improving the efficacy of deriving cells with a desired gene editing outcome. In conclusion, a multiplexed CRISPR/Cas9 editing strategy represents an efficient tool for the editing of complex, heterologous sequence tracts.