Figure 6.

Evaluation of nanoblades combined with rAAV6 encoding donor DNA for WAS gene locus knock-in. (A) Schematic representation of the nanoblades loaded with Cas9 and two sgRNA directed to the WAS locus (indicated before and inside the first exon) and the rAAV6 genome carrying the donor DNA for homologous recombination (HR); 3' HR and 5'HR arms are indicated. An expression cassette with GFP under control of the SFFV promoter was inserted between the HR arms. Targeted integration is represented with primer positions indicated used to confirm specific integration (WAS-Fw and GFP-Rv). (B) K562 cells were treated with rAAV6 vector alone or rAAV6 combined with MLV based nanoblades. A representative flow cytometry plot for GFP⁺ K562 cells is shown for day 3, 5, 7, and 10 post-treatment (left). A summary of the results is shown in the graph on the right (means ± SD; n = 4; student t-test, **p < 0.01, ***p < 0.001). MOIs for rAAV are indicated. (C) K562 genomic DNA at day 12 post-treatment was isolated and integration was determined by PCR using WAS-Fw and GFP-Rv indicated in A. The gel is representative of n = 4.