Predictable CRISPR/Cas9-Mediated COL7A1 Reframing for Dystrophic Epidermolysis Bullosa

The efficacy and the mutation spectrum of genome editing methods can vary substantially depending on the targeted sequence. A simple, quick assay to accurately characterize and quantify the induced mutations is therefore needed. Here we present TIDE, a method for this purpose that requires only a pair of PCR reactions and two standard capillary sequencing runs. The sequence traces are then analyzed by a specially developed decomposition algorithm that identifies the major induced mutations in the projected editing site and accurately determines their frequency in a cell population. This method is cost-effective and quick, and it provides much more detailed information than current enzyme-based assays. An interactive web tool for automated decomposition of the sequence traces is available. TIDE greatly facilitates the testing and rational design of genome editing strategies. Show more

In mammalian cells, DNA double-strand breaks (DSBs) are repaired predominantly by the non-homologous end joining (NHEJ) pathway, which includes subpathways that can repair different DNA-end configurations. Furthermore, the repair of some DNA-end configurations can be shunted to the auxiliary pathways of alternative end joining (a-EJ) or single-strand annealing (SSA). Show more

Autologous transgenic epidermal stem cell cultures are used to reconstitute almost the entire epidermis of a patient with severe junctional epidermolysis bullosa.