In vitroandIn vivoGenetic Disease Modelling via NHEJ-precise deletions using CRISPR/Cas9

Abstract

49Development of advanced gene and cell therapies for the treatment of genetic diseases requires 50 confident animal and cellular models to test their efficacy and is crucial in the cases where no 51 primary samples from patients are available. CRISPR/Cas9 technology, has become one of the 52 most spread endonuclease tools for editing the genome at will. Moreover, it is known that the use 53 of two guides tends to produce the precise deletion between the guides via NHEJ. Different 54 distances between guides were tested (from 8 to 500 base pairs). We found that distances 55 between the two cutting sites and orientation of Cas9 protein-DNA interaction are important for 56 the efficiency within the optimal range (30-60 bp), we could obtain new genetically reproducible 57 models for two rare disease, a Pyruvate Kinase Deficiency model, using human primary cells, and a 58 (for in vivo primary hyperoxaluria therapeutic mouse model. We demonstrate that the use of a 2-59 guideRNAs at the optimal distance and orientation is a powerful strategy for disease modelling in 60 those diseases where the availability of primary cells is limited. 61 62 63 64 65 standard chow and water. Agxt1-/-mice were genotyped as described (Salido et al. 2006). Age-127 matched C57BL/6J mice (Harlan laboratories) were used as control animals. A final dose of 1x10 13 128 vg/kg (5x10 12 vg/kg/vector) was administered to 12-14-week-old Agxt1-/-male animals by 129 intravenous injection. Animals were sacrificed and livers harvested 21 days after the 130 administration of the vectors. 131 132 133