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Sci Rep. 2016 Nov 18;6:37540. doi: 10.1038/srep37540.

Rapid and efficient CRISPR/Cas9 gene inactivation in human neurons during human pluripotent stem cell differentiation and direct reprogramming.

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Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy.
Advanced Light and Electron Microscopy Bio-Imaging Centre, Experimental Imaging Centre, San Raffaele Scientific Institute, 20132 Milan, Italy.
Neuroengineering and medical robotics laboratory, Department of Electronics, Information and Bioengineering, Politecnico di Milano, 20133 Milan, Italy.
Avantea srl, Laboratory of Reproductive Technologies, Via Porcellasco 7f, 26100 Cremona, Fondazione Avantea, Cremona.
Neuroimmunology Unit, San Raffaele Scientific Institute, 20132 Milan, Italy.
National Research Council (CNR), Institute of Neuroscience, Milan, Italy.


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.

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