Pyruvate kinase deficiency (PKD) is an autosomal recessive disorder caused by mutations in PKLR gene that expresses the erythroid pyruvate kinase enzyme (RPK). PKD-erythrocytes suffer from energy imbalance caused by reduction of RPK activity. PKD is associated with hemolysis, reticulocytosis, splenomegaly and iron overload, and may be life-threatening. We developed a knock-in gene editing strategy by combining ribonucleoprotein electroporation and adeno-associated viral vector (rAAV6) donor delivery, getting relevant editing in hematopoietic stem and progenitor cells (HSPCs) from human mobilized peripheral blood (mPB) or human cord blood. To further measure the safety and the efficiency of this approach, we have adapted this gene editing strategy to target mouse pklr gene and test correction in vivo in a PKD mouse model. Here we described the generation of new knock-in gene editing tools, including mouse specific gRNAs (80% InDels efficiency) and AAV6 donnor templates to efficiently insert a promotor-less therapeutic human codon optimized RPK cDNA, at the genomic starting site of the mouse. Mouse progenitors and stem cells (LSK cells) obtained from the PKD mice were gene edited and transplanted into RFP-C57BL6 donor recipients. 8% edition was observed before infusion. Gene-edited PKD LSK cells engrafted efficiently and did not show anaemic symptoms. Moreover, non-corrected cells lost the engraftment over the time suggesting a selective advantage of the corrected cells. DNA barcoded vectors are now under development to investigate in vivo, in the long term, the behaviour of gene edited hematopoietic stem cells.