Journal: Nature Communications

Article Title: Targeted gene editing and near-universal cDNA insertion of CYBA and CYBB as a treatment for chronic granulomatous disease

doi: 10.1038/s41467-025-62738-2

Figure Lengend Snippet: a Schematic representation of the gene editing strategy and HDR template to correct the CYBB c.252 G > A variant. b RNP + AAV-mediated gene correction of CYBB c.252 G > A variant in heterozygous carrier peripheral blood mononuclear cells (PBMCs). c RNP + AAV-mediated gene correction of CYBB c.252 G > A variant in heterozygous carrier CD34 + HSPCs (CYBB-HSPCs) measured 4 days after nucleofection. The HDR template used for sg2-mediated correction installs a precise reversion of c.252 G > A to wild-type and thus, HDR events cannot be distinguished from wild-type alleles. d Representative plot of granulocyte differentiation as measured by the presence of CD15 + myeloid cells 14 days after initiation of differentiation of gene-edited CYBB-HSPCs. e Oxidative burst assay of granulocytes differentiated from gene-edited CYBB-HSPCs derived from a heterozygous carrier. f Schematic representation of the near-universal cDNA insertion strategies to correct CYBB variants. g The percentage of X-CGD patients potentially covered by the cDNA insertion strategy. Calculations were performed based on Roos et al. . The calculation of correctable patients does not take into account X-CGD cases caused by large deletions of part of chromosome X, such as variants found in patients with McLeod syndrome. h Targeted integration levels using the cDNA-eGFP repair template measured by GFP+ cells following granulocyte differentiation. i Targeted integration levels determined by ddPCR before and after myeloid differentiation. j Oxidative burst assay performed in granulocytes differentiated from heterozygous CYBB-HSPCs treated with sg3 RNPs and the cDNA repair template. Data represented as mean ( n = 3 biological replicates) ± standard deviation. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. Source data are provided as a Source Data file.

Article Snippet: To generate cDNA HDR repair templates, codon-diverged cDNA fragments were ordered from Twist Bioscience.

Techniques: Variant Assay, Derivative Assay, Standard Deviation

Journal: Nature Communications

Article Title: Targeted gene editing and near-universal cDNA insertion of CYBA and CYBB as a treatment for chronic granulomatous disease

doi: 10.1038/s41467-025-62738-2

Figure Lengend Snippet: a Off-target sites of CYBB sg3 nominated by combining the top 10 in silico predicted off-target sites with DISCOVER-seq hits. DISCOVER-seq was carried out using both standard (STD) Cas9 and HiFi Cas9. b High-throughput amplicon sequencing of nominated off-target sites. c Semi-solid methylcellulose-based CFU assay of gene-edited CYBB-HSPCs. d Human chimerism in peripheral blood, spleen, and bone marrow of NOG mice 16 weeks after injection. e Distribution of human CD33 + myeloid cells and CD19 + B-cells in the peripheral blood of NOG mice 16 weeks after injection. f Variant frequencies determined by sequencing of the human graft in the bone marrow (BM) of NOG mice, as well as of the injected CYBB-HSPCs (input). g The percentage of GFP + cells within the human graft in the peripheral blood of NOG mice 16 weeks after injection. h ddPCR-based quantification of cDNA integration in the input CYBB-HSPCs and within the human graft in the bone marrow. Data represented as mean ( n = 3 biological replicates) ± standard deviation. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. Source data are provided as a Source Data file.

Article Snippet: To generate cDNA HDR repair templates, codon-diverged cDNA fragments were ordered from Twist Bioscience.

Techniques: In Silico, High Throughput Screening Assay, Amplification, Sequencing, Colony-forming Unit Assay, Injection, Variant Assay, Standard Deviation