I-SceI-Mediated Double-Strand Break Does Not Increase the Frequency of Homologous Recombination at the Dct Locus in Mouse Embryonic Stem Cells

Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase (Dct) is specifically expressed in melanocytes and their precursors. To construct a genetic tool allowing the replacement of Dct gene by any gene of interest, we generated an embryonic stem cell line carrying the recognition site for the yeast I-SceI meganuclease embedded in the Dct genomic segment. The embryonic stem cell line was electroporated with an I-SceI expression plasmid, and a template for the DSB-repair process that carried sequence homologies to the Dct target. The I-SceI meganuclease was indeed able to introduce a DSB at the Dct locus in live embryonic stem cells. However, the level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I-SceI to mediate homologous recombination at the Dct locus. These data suggest that homologous recombination by meganuclease-induced DSB may be locus dependent in mammalian cells

Human OTULIN haploinsufficiency impairs cell-intrinsic immunity to staphylococcal alpha-toxin

The molecular basis of interindividual clinical variability upon infection with Staphylococcus aureus is unclear. We describe patients with haploinsufficiency for the linear deubiquitinase OTULIN, encoded by a gene on chromosome 5p. Patients suffer from episodes of life-threatening necrosis, typically triggered by S. aureus infection. The disorder is phenocopied in patients with the 5p- (Cri-du-Chat) chromosomal deletion syndrome. OTULIN haploinsufficiency causes an accumulation of linear ubiquitin in dermal fibroblasts, but tumor necrosis factor receptor-mediated nuclear factor kappa B signaling remains intact. Blood leukocyte subsets are unaffected. The OTULIN-dependent accumulation of caveolin-1 in dermal fibroblasts, but not leukocytes, facilitates the cytotoxic damage inflicted by the staphylococcal virulence factor alpha-toxin. Naturally elicited antibodies against alpha-toxin contribute to incomplete clinical penetrance. Human OTULIN haploinsufficiency underlies life-threatening staphylococcal disease by disrupting cell-intrinsic immunity to alpha-toxin in nonleukocytic cells.Peer reviewe

Crispr/Cas9-based COL7A1 editing for recessive dystrophic epidermolysis bullosa

Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare and severe genetic skin disease responsible for blistering of the skin and mucosa. RDEB is caused by a wide variety of mutations in COL7A1 encoding type VII collagen, the major component of anchoring fibrils which are key attachment structures for dermal- epidermal adhesion. Site-specific CRISPR/Cas9-mediated Homo- logous Recombination (HR) is emerging as a powerful approach for gene editing to correct disease mutations. Here we provide preliminary data for COL7A1 editing using the CRISPR/Cas9 approach without selection. We designed five guide RNAs (gRNAs) in order to correct a RDEB causative null mutation in exon 2 (c.189delG; p.Lys6Trp*40). Four of them showed efficient expression, low toxicity and up to 20% of activity in HEK293 cells. These gRNAs were cloned into a lentiviral vector and delivered as integration-deficient lentivirus (IDLVs). Among the four site-spe- cific gRNAs tested, two showed significant activity in HEK293 cells (up to 35%), in primary keratinocytes and in fibroblasts (up to 15%) when delivered as IDLVs. To achieve gene editing in RDEB cells, primary keratinocytes and fibroblasts isolated from a RDEB patient homozygous for the c.189delG mutation were co-trans- duced with IDLVs encoding the site-specific CRISPR/Cas9 and the corresponding Donor. Genetic correction could be detected in bulk- transduced cells when performing allele-specific PCR and direct sequencing. In addition, type VII collagen was detected in up to 10% of these cells as assessed by immunochemistry. Next steps will aim at improving the efficiency of CRISPR/Cas9-mediated HR for the development of transplantable skin models suitable for clinical application

Ex Vivo COL7A1 Correction for Recessive Dystrophic Epidermolysis Bullosa Using CRISPR/Cas9 and Homology-Directed Repair

International audienceRecessive dystrophic epidermolysis bullosa is a rare and severe genetic skin disease resulting in blistering of the skin and mucosa. Recessive dystrophic epidermolysis bullosa (RDEB) is caused by a wide variety of mutations in COL7A1-encoding type VII collagen, which is essential for dermal-epidermal adhesion. Here we demonstrate the feasibility of ex vivo COL7A1 editing in primary RDEB cells and in grafted 3D skin equivalents through CRISPR/Cas9-mediated homology-directed repair. We designed five guide RNAs to correct a RDEB causative null mutation in exon 2 (c.189delG; p.Leu64Trpfs*40). Among the site-specific guide RNAs tested, one showed significant cleavage activity in primary RDEB keratinocytes and in fibroblasts when delivered as integration-deficient lentivirus. Genetic correction was detected in transduced keratinocytes and fibroblasts by allele-specific highly sensitive TaqMan-droplet digital PCR (ddPCR), resulting in 11% and 15.7% of corrected COL7A1 mRNA expression, respectively, without antibiotic selection. Grafting of genetically corrected 3D skin equivalents onto nude mice showed up to 26% re-expression and normal localization of type VII collagen as well as anchoring fibril formation at the dermal-epidermal junction. Our study provides evidence that precise genome editing in primary RDEB cells is a relevant strategy to genetically correct COL7A1 mutations for the development of future ex vivo clinical applications

Different expression of synemin isoforms in glia and neurons during nervous system development

International audienc