Enhancing CRISPR/Cas9-mediated homology-directed repair in mammalian cells by expressing Saccharomyces cerevisiae Rad52 |
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| Affiliation: | 1. College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China;2. Guangzhou Key Laboratory of Insect Development Regulation and Application Research, School of Life Sciences, South China Normal University, Guangzhou 510631, China;3. Institute of Lung and Molecular Therapy, Xinxiang Medical University, Xinxiang, Henan 453003, China;1. The Gladstone Institute of Cardiovascular Disease, 1650 Owens Street, San Francisco, CA 94158, USA;2. Department of Bioengineering, Stanford University, Stanford, CA 94305, USA;3. Bioinformatics Division, Center for Synthetic and Systems Biology, TNLIST/Department of Automation, Tsinghua University, Beijing 100084, China;6. Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA;7. ChEM-H, Stanford University, Stanford, CA 94305, USA;1. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark;2. Joint BioEnergy Institute, Emeryville, CA, USA;3. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA;4. Department of Chemical and Biomolecular Engineering & Department of Bioengineering University of California, Berkeley, CA, USA;1. Department of Biomedicine, Aarhus University, Aarhus, Denmark;2. University of Cambridge, UK;3. Max-Delbrück-Center for Molecular Medicine, 13125 Berlin, Germany;4. Berlin Institute of Health, 10117 Berlin, Germany;1. Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA;2. Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, 02454-9110, USA |
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| Abstract: | Precise genome editing with desired point mutations can be generated by CRISPR/Cas9-mediated homology-directed repair (HDR) and is of great significance for gene function study, gene therapy and animal breeding. However, HDR efficiency is inherently low and improvements are necessitated. Herein, we determined that the HDR efficiency could be enhanced by expressing Rad52, a gene that is involved in the homologous recombination process. Both the Rad52 co-expression and Rad52-Cas9 fusion strategies yielded approximately 3-fold increase in HDR during the surrogate reporter assays in human HEK293T cells, as well as in the genome editing assays. Moreover, the enhancement effects of the Rad52-Cas9 fusion on HDR mediated by different (plasmid, PCR and ssDNA) donor templates were confirmed. We found that the HDR efficiency could be significantly improved to about 40% by the combined usage of Rad52 and Scr7. In addition, we also applied the fusion strategy for modifying the IGF2 gene of porcine PK15 cells, which further demonstrated a 2.2-fold increase in HDR frequency. In conclusion, our data suggests that Rad52-Cas9 fusion is a good option for enhancing CRISPR/Cas9-mediated HDR, which may be of use in future studies involving precise genome editing. |
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| Keywords: | CRISPR/Cas9 Rad52 Homology-directed repair Surrogate reporter Precise genome editing |
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