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 language | English |
|---|---|
| Article number | 37540 |
| Journal | Scientific Reports |
| Volume | 6 |
| DOIs | |
| Publication status | Published - Nov 18 2016 |
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Rapid and efficient CRISPR/Cas9 gene inactivation in human neurons during human pluripotent stem cell differentiation and direct reprogramming. / Rubio, Alicia; Luoni, Mirko; Giannelli, Serena G.; Radice, Isabella; Iannielli, Angelo; Cancellieri, Cinzia; Di Berardino, Claudia; Regalia, Giulia; Lazzari, Giovanna; Menegon, Andrea Domenico; Taverna, Stefano; Broccoli, Vania.
In: Scientific Reports, Vol. 6, 37540, 18.11.2016.Research output: Contribution to journal › Article
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TY - JOUR
T1 - Rapid and efficient CRISPR/Cas9 gene inactivation in human neurons during human pluripotent stem cell differentiation and direct reprogramming
AU - Rubio, Alicia
AU - Luoni, Mirko
AU - Giannelli, Serena G.
AU - Radice, Isabella
AU - Iannielli, Angelo
AU - Cancellieri, Cinzia
AU - Di Berardino, Claudia
AU - Regalia, Giulia
AU - Lazzari, Giovanna
AU - Menegon, Andrea Domenico
AU - Taverna, Stefano
AU - Broccoli, Vania
PY - 2016/11/18
Y1 - 2016/11/18
N2 - 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.
AB - 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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U2 - 10.1038/srep37540
DO - 10.1038/srep37540
M3 - Article
VL - 6
JO - Scientific Reports
JF - Scientific Reports
SN - 2045-2322
M1 - 37540
ER -