Fig. 5: CRISPR/Cas9 targeting of a putative enhancer element in the SIX2-SIX3 locus in primary human islets.

A Genome Browser tracks of the genomic context in the SIX2-SIX3 locus, highlighting the putative SIX2-SIX3 enhancer element (SIXE) with variants previously linked to increased risk of fasting glucose hyperglycemia and T2D (T2D-SNPs, black arrowheads): These variants map to an active enhancer within an enhancer cluster (yellow line, and zoomed inset); sgRNAs used for CRISPR-Cas9 targeting of the region flanking FG-SNP: SIXE-3′ and SIXE-5′ (red arrows). The two sgRNAs were cloned in the same construct (scheme in Fig. 4A). Chromatin classes: active promoter (green); active enhancer (red); inactive enhancer (gray); inactive open chromatin (black); strong CTCF (blue). Accessible chromatin regions in human islets are shown by ATAC-seq, H3K27ac and mediator ChiP-seq. B Islet eQTLs showing association of reduced expression levels of SIX2 and SIX3 and variants within the SIXE region targeted in this study (cis-eQTL mapping across 292 human islet samples, q-val= 4.9e−12 for rs12712929-TT and reduced level of SIX3 and q-val= 0.008, for rs12712928 -CC and reduced expression of SIX2). Box plot shows the interquartile range (IQR) of 1st (Q25) and 3rd (Q75) quartiles, with the median as a black line in the center, and whiskers depict ± 1.5 times the IQR. For SIX3 and GT, GT, and TT genotypes, Q25 values are 2.09, 1.41 and −2.03, and Q75 values are 3.92, 3.04, and −1.10, respectively. For SIX2 and GG, GC and CC genotypes, Q25 values are 2.02, 1.89 and 1.27, and Q75 values are 3.42, 3.20, and 1.03, respectively. C Scheme of the ddPCR assay used to identify gene editing as consequence of targeting by SIXE-5′, SIXE-3′, or both (SIXE-5′−3′) in the pseudoislets targeted with SIXE-5′–3′. HEX probes are shown with an orange line, FAM reference probes, in blue D–F Example of ddPCR 2D plots showing GFP⁺ cells modified by (D) SIXE-5′, (E) SIXE-3′, (F) SIXE-5′–3′ (KO cells: FAM⁺/HEX⁻, blue droplets; wild-type droplets: FAM⁺/HEX⁺, orange, n = 2 independent donor samples). G Total percentage of gene-edited alleles in GFP⁺ cells, showing the contribution of (D–F) in the DNA of GFP⁺ islet cells. H–J mRNA levels of (H) SIX3 (n = 6 independent donors; P = 0.0279), SIX3-AS1 (n = 5 independent donors; P = 0.0437) and SIX2 (n = 6 independent donors; P = 0.04), (I) control genes (n = 4 independent donor samples), (J) INS (n = 4 independent donor samples) in GFP⁺ cells targeted with CRISPR-SIXE-5′–3′. (K) Total insulin content of sorted GFP⁺ cells normalized to genomic DNA (gDNA) content (P = 0.0391; n = 4 independent donor samples). Data are presented as mean values ± SD. Two-tailed t tests were used to generate P-values. *P < 0.05. Source data are provided as a Source Data file.