Fig. 5: Validation of causality between differentially expressed genes and 22q11.2del in an isogenic setting.

a Generation of isogenic lines with 22q11.2del using CRISPR/Cas9 guide RNAs that cut within the low copy repeats (LCRs) flanking the 3 Mb deletion. Coordinates for the guides genomic position on chromosome 22 are indicated (Hg19). b Detection of isogenic 22q11.2del using DNA FISH analysis and a probe generated probe using CTD-2300P14 (Thermo Fisher Scientific, 96012). Blue = DAPI (DNA), Red=22q11.2 region. Scale bar: 10um. (N(edited clones) = 2 and N(nonedited clones) = 2; 3 experimental replicates each). c ddPCR assay to determine the copy numbers of HIRA and ZNF74, located in 22q11.2 (N=2 wildtype and 2 edited clones and 1 patient control). Analysis performed via QuantaSoft software (BioRad); copy number for HIRA and ZNF74 (normalized to RRP30), error bars represent the Poisson 95% confidence limits. d SNP array marker intensity (LRR) for SNPs overlapping the deletion locus confirms 22q11.2del in two clones (red). e,f Principal component analysis of cell lines with and without isogenic 22q11.2del. Circles = genes within 22q11.2 (cis). Triangles = genes outside 22q11.2 (trans). e PC1 and PC2 separate cells by developmental stage., PC3 and PC4 separate cells by deletion genotype. g Significant downregulation of genes in 22q11.2 in lines with isogenic 22q11.2del (Mann-Whitney U test for 32 genes in 2 deletion and 2 control clones, two-sided). Data is presented in a Tukey-style boxplot with the median (Q2) and the first and the second quartiles (Q2, Q3) and error bars defined by the last data point within +/− 1.5-times the interquartile range. h Correlation of fold changes in differentially expressed genes in discovery and isogenic datasets in neurons. Transcripts from 32 genes were detected and significantly changed in the discovery and isogenic lines (adjusted p-value < 0.05), of which nine were located outside 22q11.2 (FAM13B, KMT2C, HYAL2, DNPH1, ZMYM2, VAPB, SMG1, CPSF4, MAP3K2). All 32 genes were changed in the same direction in both cohorts (p = 5.6 × 10⁻⁹, binomial test). Genes with a SynGO annotation shown in red, genes with no SynGO annotation shown in blue. Circles = cis genes. Triangles = trans genes. i, MEF2C is upregulated in 22q11.2del NPCs compared to isogenic controls (N(edited clones) = 2 and N(non-edited clones) = 2; 3 experimental replicates each). Data is presented in a Tukey style boxplot with the median (Q2) and the first and the second quartiles (Q2, Q3) and error bars defined by the last data point within +/− 1.5-times the interquartile range. j SynGo annotation of genes induced in isogenic 22q11.2del neurons showing enrichment for synaptic vesicle cycle and endocytosis.