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

Alicia Rubio, Mirko Luoni, Serena G. Giannelli, Isabella Radice, Angelo Iannielli, Cinzia Cancellieri, Claudia Di Berardino, Giulia Regalia, Giovanna Lazzari, Andrea Domenico Menegon, Stefano Taverna, Vania Broccoli

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Abstract

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.

Original languageEnglish
Article number37540
JournalScientific Reports
Volume6
DOIs
Publication statusPublished - Nov 18 2016

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Rubio, A., Luoni, M., Giannelli, S. G., Radice, I., Iannielli, A., Cancellieri, C., Di Berardino, C., Regalia, G., Lazzari, G., Menegon, A. D., Taverna, S., & Broccoli, V. (2016). Rapid and efficient CRISPR/Cas9 gene inactivation in human neurons during human pluripotent stem cell differentiation and direct reprogramming. Scientific Reports, 6, [37540]. https://doi.org/10.1038/srep37540