Genome editing (GE) technologies based on CRISPR/Cas systems allow targeted genomic modification and have emerged as powerful alternatives to conventional gene addition for various human diseases, with a series of clinical trials in progress. However, some challenges remain to be addressed to obtain more effective precise sequence changes. Novel approaches, such as prime editors (PEs) have raised exciting possibilities, allowing double-strands break-free GE. However, a major limitation of PEs is highly variable efficiency both from one target to another and between cell types. In addition, PEs can only make small sequence changes. Our objectives are to develop (i) alternative, more efficient and safer GE tools based on the PE strategy We will design and evaluate novel PE tools in order to both increase activity per se and overcome cell-specific limitations. We will translate the novel PE tools in an in vivo vertebrate system, the zebrafish, chosen for its amenability to high throughput screening and ease of manipulation. (ii) universal gene therapy strategies for Duchenne Muscular Dystrophy (DMD) and ?-hemoglobinopathies taking advantage of the power of the novel GE tools DMD is the most common form of muscular dystrophy and is caused by mutations in the Dystrophin gene. There is still no effective treatment and a promising universal therapeutic strategy consists in upregulating expression of utrophin in skeletal muscle. We will use PE to upregulate Utrophin by disrupting repressor binding sites. ?-thalassemia and Sickle Cell Disease are frequent genetic diseases caused by mutations in the ?-globin locus. Importantly, clinical severity is alleviated in patients with genetic variants causing hereditary persistence of fetal hemoglobin (HPFH). A universal therapeutic strategy thus aims at generating HPFH mutations in hematopoietic stem/progenitor cells. We propose to use PE to introduce multiple mutations in one step to better rescue the ?-hemoglobinopathy phenotype.