Rapid and efficient CRISPR/Cas9 gene inactivation in human neurons during human pluripotent stem cell differentiation and direct reprogramming
| Autor: | Andrea Menegon, Vania Broccoli, Giulia Regalia, Angelo Iannielli, Cinzia Cancellieri, Claudia Di Berardino, Mirko Luoni, Isabella Radice, Giovanna Lazzari, Stefano Taverna, Serena Giannelli, Alicia Rubio |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Cellular differentiation Genetic Vectors Induced Pluripotent Stem Cells Biology Genome Article 03 medical and health sciences Neural Stem Cells Genome editing Humans CRISPR Gene Silencing Induced pluripotent stem cell Neurons Genetics Multidisciplinary Cas9 Gene targeting Cell Differentiation Fibroblasts Cellular Reprogramming Cell biology 030104 developmental biology CRISPR-Cas Systems Reprogramming |
| Zdroj: | Scientific Reports |
| ISSN: | 2045-2322 |
| Popis: | The CRISPR/Cas9 system is a rapid and customizable tool for gene editing in mammalian cells. In particular, this approach has widely opened new opportunities for genetic studies in neurological disease. Human neurons can be differentiated in vitro from hPSC (human Pluripotent Stem Cells), hNPCs (human Neural Precursor Cells) or even directly reprogrammed from fibroblasts. Here, we described a new platform which enables, rapid and efficient CRISPR/Cas9-mediated genome targeting simultaneously with three different paradigms for in vitro generation of neurons. This system was employed to inactivate two genes associated with neurological disorder (TSC2 and KCNQ2) and achieved up to 85% efficiency of gene targeting in the differentiated cells. In particular, we devised a protocol that, combining the expression of the CRISPR components with neurogenic factors, generated functional human neurons highly enriched for the desired genome modification in only 5 weeks. This new approach is easy, fast and that does not require the generation of stable isogenic clones, practice that is time consuming and for some genes not feasible. |
| Databáze: | OpenAIRE |
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