title: Rapid and efficient CRISPR/Cas9 gene inactivation in human neurons during human pluripotent stem cell differentiation and direct reprogramming.
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.
authors.affiliation: Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy.
authors.forename: Alicia
authors.initials: A
authors.lastname: Rubio
authors.affiliation: Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy.
authors.forename: Mirko
authors.initials: M
authors.lastname: Luoni
authors.affiliation: Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy.
authors.forename: Serena G
authors.initials: SG
authors.lastname: Giannelli
authors.affiliation: Advanced Light and Electron Microscopy Bio-Imaging Centre, Experimental Imaging Centre, San Raffaele Scientific Institute, 20132 Milan, Italy.
authors.forename: Isabella
authors.initials: I
authors.lastname: Radice
authors.affiliation: Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Iannielli
authors.affiliation: Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Cancellieri
authors.affiliation: Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Di Berardino
authors.affiliation: 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.
authors.forename: Giulia
authors.initials: G
authors.lastname: Regalia
authors.affiliation: Avantea srl, Laboratory of Reproductive Technologies, Via Porcellasco 7f, 26100 Cremona, Fondazione Avantea, Cremona.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Lazzari
authors.affiliation: Advanced Light and Electron Microscopy Bio-Imaging Centre, Experimental Imaging Centre, San Raffaele Scientific Institute, 20132 Milan, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Menegon
authors.affiliation: Neuroimmunology Unit, San Raffaele Scientific Institute, 20132 Milan, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Taverna
authors.affiliation: Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy.|National Research Council (CNR), Institute of Neuroscience, Milan, Italy.
authors.forename: Vania
authors.initials: V
authors.lastname: Broccoli
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics*; D065150:Cellular Reprogramming / Q000235:genetics*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D020868:Gene Silencing; D005822:Genetic Vectors; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D058953:Neural Stem Cells / Q000145:classification* / Q000166:cytology / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism