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. 2021 Aug 13;12:4922. doi: 10.1038/s41467-021-25190-6

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a CGH/SNP-array: Log₂Ratio and BAF (B-allele frequency) in control-HSPC cells and two edited LOH-positive-clones, zoomed on Chr11p15. Globin cut-site location is indicated by a black line. RRM1 and CARS SNP losses are indicated by a blue line. LOH are framed. b Percentages of unmethylated and methylated alleles of H19 differentially methylated region (H19 DMR), H19 gene proximal promoter and KCNQ1OT1 (KCNQ1 opposite strand/antisense transcript 1) in NT-CD34+ (non-transduced parental CD34+ cells), in five edited clones without LOH (34.11, 34.13, 34.33, 34.36, 34.80), in a pool of negative LOH edited clones, in 3 clones with LOH (34.8, 34.15 and 34.40) analyzed by quantitative real-time methylation-specific PCR (qMSP). in vitro methylated DNA and No DNA (NTC) were used as positive and negative controls. ND: non determined because of lack of DNA cell material. c Diagram of Chr11p15.4-p15.5 band with position of imprinted genes and CRISPR-Cas9-induced DNA-DSB (red arrow) in HSPCs. Paternal and maternal alleles in light blue and pink, respectively. Unmethylated and methylated CpG islands represented by white and black circles. Genes expressed from parental allele in dark blue, gene silenced on parental allele in red. Normal paternal and maternal allele from NT-CD34+ cells and alleles from paternal and maternal iCN-LOH clones are represented. d H19 and IGF2 mRNA expression in NT-CD34+ cells, in four edited LOH-negative clones (34.4, 34.17, 34.80, and 34.96) and two LOH-positive clones (34.8 and 34.15). Results are normalized using TATA-box binding protein (TBP) housekeeping gene. LOD, limit of detection. Quantification is represented by mean and SEM from the same sample measured repeatedly. H19 mRNA is not or hardly detectable in LOH-positive clones. IGF2 mRNA is only detectable in LOH-positive clones. Source data are provided as a Source Data file.