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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. 1 — a Experimental design. HEK293T cells edited with Cas9 nuclease RNP and sets of gRNA corresponding to protocols #1 to #3. Map of β-globin genes, telomeric downstream SNP genes (RRM1) and FISH probes. LOH, loss of heterozygosity; SNP, single-nucleotide polymorphism; FISH, fluorescent in situ hybridization; CGH, comparative genomic hybridization. b Illustrative Chr11 DNA-FISH in HEK cells. Scale-bar: 5 µm. Polyclonal Chr11 DNA-FISH analysis at D4 (left) and at D18 (right) after editing with protocols #1 to #3. NT: non-transfected. O, Orange sub-centromeric probe; G sub-telomeric Green probe. 3O/3G (in gray), 3O/2G (in red), 2O/3G (in yellow), and 2O/2G (in black) signal percentages are quantified. 3O/2G frequencies after protocols 1–3 were compared to non-transduced cells on same day by two-sided Chi-square tests. n correspond to analyzed cells from each polyclonal pool (1 per protocol). c left: Polyclonal Chr11 DNA-FISH analysis at D4 after editing with protocol #3 (Chr11), without gRNA or targeting another chromosome (UROS locus in Chr10). 3O/2G frequencies targeting Chr10 or without guide RNA were compared to protocol #3 on same day. 3O/2G frequencies targeting Chr10 or without guide RNA were compared by two-sided Chi-square tests. Right: Polyclonal Chr11 DNA-FISH analysis at D4 after editing with protocol #3 (Chr11), and with protocol #3 using HiFi Cas9. 3O/3G, 3O/3G, 2O/3G and 2O/2G signal percentages were quantified. 3O/2G frequencies with protocol #3 using HiFi Cas9 or Cas9 were compared to non-transduced cells by two-sided Chi-square tests. 3O/2G frequencies with protocol #3 using HiFi Cas9 were compared to protocol #3 using Cas9 by two-sided Chi-square tests. O: orange sub-centromeric Chr11 probe, G: green sub-telomeric Chr11p probe. n correspond to analyzed cells from each polyclonal pool (1 per protocol). Source data are provided as a Source Data file.