Design of rAAV6-MAGT1 donor for CRISPR/Cas9 targeted integration into XMEN CD34⁺ HSPCs. (A) Location of the 10-candidate sgRNAs targeting MAGT1 gene transcription start site. Each sgRNA (sg) is represented as a 20-bp sequence (light pink or green) associated with the 3-bp protospacer adjacent motif sequence (darker color). (B) Evaluation of the percentage of cutting activity by tracking of indels by decomposition (TIDE) assay of indel formation after guide DNA (gDNA) extraction from untreated vs Cas9/sgRNA-treated HD CD34⁺ cells at 5 days post-electroporation (EP), sequencing of exon 1, and comparison of the sequences using TIDE software (data represent 1-5 independent experiments ). (C) Design of the rAAV6-MAGT1 donor. (D) Molecular analysis showing the percentage of TIs in gene-edited CD34⁺ cells (3 different donors, 2 HDs and XMEN patient 1) using GE enhancers as described. gDNA was extracted at 5 days post-EP, and insertion of the MAGT1 cDNA donor was quantified by droplet digital PCR (ddPCR) (4 independent experiments for –i53, 7 for +i53, and 7 for +i53+hGSE). (E) Viability was determined by trypan blue exclusion in naive and rAAV6-MAGT1–treated HSPCs at day 2 post-EP (5 independent experiments for –i53, 10 for +i53, and 10 for +i53+hGSE). (F) Bar graphs of HSPC subpopulations (multilymphoid progenitor [MLP], CD34⁺CD38⁺CD45RA^–; common myeloid progenitor [CMP], CD34⁺CD38⁺CD45RA^–; HSCs, CD34⁺CD38^–CD45RA^–CD90⁺CD133⁺; and multipotential progenitor [MPP], CD34⁺CD38^–CD45RA^–CD90^–]) 2 days EP (data represent 4 independent experiments for –i53, 4 for +i53, and 7 for +i53+hGSE), compared with naive cells. Data are shown as mean ± standard deviation (SD); analysis of variance (ANOVA) 1-way test and Tukey’s post hoc multiple comparisons test were used. *P < .05; **P < .01; ***P < .001; ****P < .0001. Fwd, forward; ITR, inverted terminal repeat; ns, nonsignificant; LHA, left homology arm; polyA, poly(A) tail; RHA, right homology arm; Rev, reverse; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element.