Precise editing for ADA2 deficiency

CRISPR/Cas9 technology has been shown to be able to correct harmful mutations in variety of human cells and enable numerous clinical trials to be started already. Here, we took advantage of convenience and elegance of this editing system and introduced model SNPs into ADA2 mutation specific region in human CD34+ cord blood haematopoietic stem and progenitor cells (HSPCs). First, we screened 7 sgRNAs together with ssODN repair templates spanning C→T (R169Q) ADA2 mutation in HSPCs. We validated the editing outcomes by Next generation amplicon sequencing, Digital-Droplet PCR (ddPCR) and ICE analysis. To ensure the edited cells with the best performing sgRNA were fully functional, we utilized standard NSG mouse model NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (Charles Rivers) for xenotransplantation study including optimized irradiation protocol. Finally, we did not observe suppression of engraftment levels and were able to detect HDR events in subpopulations of HSPCs and differentiated immune cells after 12 weeks post intravenous injection. Nevertheless we saw donor to donor variation in editing and engraftment potential. Additionally, we tested chemically modified ssODNs and DNA repair interfering factors GSE56, Ad5 E4 Orf6&7 and i53 and secured HDR improvement in HSPCs in vitro. Taken together, we presented workflow and evidence for future application of precise and tailored approach for editing and engrafting of ADA2-SCID patients derived CD34+ cells.