Articles by Daniela Benati in JoVE
An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector Daniela Benati1, Fabienne Cocchiarella1, Alessandra Recchia1 1Centre for Regenerative Medicine, Department of Life Sciences, University of Modena and Reggio Emilia This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.
Other articles by Daniela Benati on PubMed
In Vivo Editing of the Human Mutant Rhodopsin Gene by Electroporation of Plasmid-based CRISPR/Cas9 in the Mouse Retina Molecular Therapy. Nucleic Acids. Nov, 2016 | Pubmed ID: 27874856 The bacterial CRISPR/Cas system has proven to be an efficient tool for genetic manipulation in various organisms. Here we show the application of CRISPR-Cas9 technology to edit the human Rhodopsin (RHO) gene in a mouse model for autosomal dominant Retinitis Pigmentosa. We designed single or double sgRNAs to knock-down mutant RHO expression by targeting exon 1 of the RHO gene carrying the P23H dominant mutation. By delivering Cas9 and sgRNAs in a single plasmid we induced an efficient gene editing in vitro, in HeLa cells engineered to constitutively express the P23H mutant RHO allele. Similarly, after subretinal electroporation of the CRISPR/Cas9 plasmid expressing two sgRNAs into P23H RHO transgenic mice, we scored specific gene editing as well as significant reduction of the mutant RHO protein. Successful in vivo application of the CRISPR/Cas9 system confirms its efficacy as a genetic engineering tool in photoreceptor cells.