Fig. 3: Genotoxicity analysis using high-quality SNP-amplicons revealed that HBG1/2 promoter targeting by CRISPR induces a wide range of LOHs in HSPCs. | Nature Communications

Fig. 3: Genotoxicity analysis using high-quality SNP-amplicons revealed that HBG1/2 promoter targeting by CRISPR induces a wide range of LOHs in HSPCs.

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

Fig. 3: Genotoxicity analysis using high-quality SNP-amplicons revealed that HBG1/2 promoter targeting by CRISPR induces a wide range of LOHs in HSPCs.

a Experimental design. HSPCs from cord blood (CB) were transfected with Cas9 nuclease RNP and gRNA targeting HBG1/2 promoters and scSNP-DNAseq was performed at day 4. Five high-quality SNPs along the Chr11 were chosen (megabasic SNP panel), SNP1 on the non-targeted region 11q, SNP2 in Chr11p between the centromere and the cut site HBG1/2p and SNP3-5 telomeric to the cut site at 0.3, 2.8, and 4.6 Mb. b Histogram represents the cell percentages with LOH at day 4 (n = 1870 cells). Presence of 3 LOH profiles (WT = no LOH, telomeric LOH11pI and LOH11pII with loss of SNP3-5 (red or blue allele respectively). c Description of the Megabasic SNP panel and the different SNP profiles identified by sc-SNP-DNA seq (WT, LOH 11pI, LOH 11pII). d A novel set of SNPs was chosen using two interstitial SNPs (6 and 7) on the telomeric side (kilobasic SNP panel). e scSNP-DNAseq and kilobasic SNP panel pipeline revealed the presence of 2 novel interstitial LOH profiles (iLOH). Histogram represents the % of cells with interstitial and telomeric LOH (iLOH and tLOH) at day 4 (n = 1855 cells). f Cumulative histogram of copy-neutral and copy-loss LOHs in each profile (number of cells indicated). g Ploidies and cGH-like representations (CNV analyses) along the targeted Chr11 for the 5 profiles (WT, LOH11pI, LOH 11pII, iLOH11pI, and iLOH11pII), and of the non-targeted Chr10 (negative control). Ploidy in LOH genomic area was calculated by the mean ratio of read depth of amplicons in the LOH area of edited LOH cells to non-edited cells. h Different SNP profiles identified by sc-SNP-DNA seq and kilobasic SNP panel pipeline (WT = no LOH), LOH 11pI, LOH 11pII, iLOH 11pI, iLOH 11pII). Created in BioRender. Bedel, A. (2025) https://BioRender.com/3fwjwd9.

Back to article page