Fig. 4: Time course of AZD7648 short-term megabase-scale genotoxicity in hFFs and HSPCs. | Nature Communications

Fig. 4: Time course of AZD7648 short-term megabase-scale genotoxicity in hFFs and HSPCs.

From: Single-cell multiplex approaches deeply map ON-target CRISPR-genotoxicity and reveal its mitigation by palbociclib and long-term engraftment

Fig. 4: Time course of AZD7648 short-term megabase-scale genotoxicity in hFFs and HSPCs.

a Experimental design. hFFs were transfected with Cas9 RNP, ssODN template and gRNA targeting ABRAXAS2, with or without HDR boosters (AZD7648, red: 1 µM; NU7441, orange: 2.5 µM; XL413, green: 20 µM; DMSO control, blue). Schema of the Chr10 and analyzed SNPs. Cell cycle phases (day 2), micronuclei count (day 4), editing efficacy (day 7), LOH detection by FAMReD (day 18), and scSNP-DNAseq kinetic were performed. b Left panel, % of micronuclei (MN) quantified by cytometry (Mean ± SD, n = 5 independent experiments, One-way ANOVA test (two-tailed)). Right panel, % of fluorescent cells induced by ABRAXAS2 editing at day 18 using the FAMReD system. Fluorescent cells are due to telomeric megabase-scale LOH (Mean ± SD, n = 4 independent experiments, One-way ANOVA test (two-tailed)). c 5 high-quality SNPs along the Chr10, present in unedited cells, were selected. % of LOH in hFFs detected by scSNP-DNAseq at day 2, 7, and 20, with or without CRISPR-Cas9 and AZD7648 (right histogram with AZD7648, Chi-square test (two-tailed)). Number of cells exhibiting LOH10pI and LOH10pII profiles, ploidy, and % of copy-loss LOH. d Experimental design. HSPCs were transfected with Cas9 RNP, ssODN template, and gRNA targeting UROS, with/without AZD7648. Schema of the Chr10 and analyzed SNPs. Micronuclei count (day 2), editing efficacy (day 7), and scSNP-DNAseq kinetic were performed. e Histogram with % of MN after HSPCs editing (Mean ± SD, n = 3 independent experiments, One-way ANOVA test (two-tailed)). f 5 high-quality SNPs along the Chr10, identified in unedited cells, were selected (megabasic panel). % of LOH in HSPCs by scSNP-DNAseq at day 4, 7, and 14 after transfection, with/without CRISPR-Cas9 and AZD7648 (right histogram, Chi-square test (two-tailed)). Number of cells exhibiting LOH10pI and LOH10pII profiles, ploidy, and % of copy-loss LOH. Created in BioRender. Bedel, A. (2025) https://BioRender.com/3fwjwd9.

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