Fig. 3: CRISPR-Cas9-mediated globin gene targeting can induce Chr11p copy-neutral-LOH (iCN-LOH) in HSPC.

a Experimental design human CD34+ from cord blood edited with Cas9 RNP and sets of gRNA corresponding to protocols #1 and #3. Map of β-globin genes, telomeric downstream SNP genes (RRM1, CARS1 and KCNQ1), telomeric imprinted genes (H19 and IGF2) and FISH probes. b Top panel illustrative Chr11 DNA-FISH in hCD34⁺ cells. Scale-bar: 5 µm. Bottom panel, polyclonal Chr11 DNA-FISH analysis at D18 of edited cells with protocols #1 (left) and #3 (right). Median of each Chr11 profile (2O/2G (in gray), 2O/1G (in green), 1O/2G (in orange)) in non-transfected cells as control (NT, Non-Transfected) and edited cells with protocols #1 and #3 (n = 7). O: orange sub-centromeric Chr11 probe, G: green sub-telomeric Chr11p probe. 2O/1G frequencies were compared between conditions by two-sided Chi-square tests. n correspond to analyzed hCD34⁺ cells for each protocol (pooled from three distinct transfections). c Polyclonal Chr2 DNA-FISH analysis at day 18 of edited cells (in Chr11) with protocol #3 or non-transfected cells. O: orange sub-telomeric Chr2 probe, G: green sub-centromeric Chr2 probe. 2G/1O (in orange) frequencies were compared between conditions by two-sided Chi-square tests. n correspond to analyzed hCD34⁺ cells for each protocol. d RRM1/CARS1 SNPs sequencing after subclonal analysis of CD34+ cells at D4, illustrating SNP-LOH observed in clones 34.8 and 34.15 from protocols #3.1 and #3.2. e Chromosome analysis of two HSPC clones with LOH by array-CGH (34.8 and 34.15). f LOH-positive clone frequencies for protocols #1, #3.1 and #3.2. Source data are provided as a Source Data file.