High-throughput assessment of mutations generated by genome editing in induced pluripotent stem cells by high-resolution melting analysis |
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| Affiliation: | 1. Liver Tissue Engineering and Cell Therapy Laboratory, CHU Sainte-Justine, Montreal, Canada;2. Pediatric Hepatology, CHU Sainte-Justine, Montreal, Canada;3. Department of Pediatrics, Université de Montréal, Montreal, Canada;1. Laboratorio de Medicina Regenerativa Cardiovascular, Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTYB), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Favaloro, Buenos Aires, Argentina;2. Laboratorio de Ingeniería Genética y Biología Celular y Molecular (LIGBCM), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional de Quilmes, Bernal, Argentina;3. Hospital Universitario de la Fundación Favaloro, Buenos Aires, Argentina;4. Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina;1. ISCT 2020 Paris Co-Chair, Institut Paoli-Calmettes, FRA;2. ISCT 2020 Paris Co-Chair, University of Verona, ITA;3. ISCT 2020 Paris Co-Chair, University Hospital Basel, CHE |
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| Abstract: | Background and aimsGenome editing of induced pluripotent stem cells (iPSCs) holds great potential for both disease modeling and regenerative medicine. Although clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 provides an efficient and precise genome editing tool, iPSCs are especially difficult to transfect, resulting in a small percentage of cells carrying the desired correction. A high-throughput method to identify edited clones is required to reduce the time and costs of such an approach.MethodsHere we assess high-resolution melting analysis (HRMA), a simple and efficient real-time polymerase chain reaction-based method, and compare it with more commonly used assays.Results and conclusionsOur data show that HRMA is a robust and highly sensitive method, allowing the cost-effective and time-saving screening of genome-edited iPSCs. Samples can be prepared directly from 96-well microtiter plates for high-throughput analysis, and amplicons can be further analyzed with downstream techniques for further confirmation, if needed. |
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