Single-cell SNP-DNA sequencing precisely maps genotoxic events in CRISPR-edited primary cells

Boutin, J.; FAYET, S.; MARIN, V.; BERGES, C.; Riandiere, M.; Toutain, J.; Lamrissi-Garcia, I.; THIBAULT, C.; Cappellen, d.; Dabernat, S.; Poulet, A.; Francilette, M.; DROUIN, N.; MOREAU-GAUDRY, F.; BEDEL, A.

doi:10.1101/2025.04.04.647179

bioRxiv · 10.1101/2025.04.04.647179

Single-cell SNP-DNA sequencing precisely maps genotoxic events in CRISPR-edited primary cells

Boutin, J.·FAYET, S.·MARIN, V.·BERGES, C.·Riandiere, M.·Toutain, J.·Lamrissi-Garcia, I.·THIBAULT, C.·Cappellen, d.·Dabernat, S.·Poulet, A.·Francilette, M.·DROUIN, N.·MOREAU-GAUDRY, F.·BEDEL, A.

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Abstract

Genome editing by CRISPR-Cas9 is promising for gene therapy. However, safety concerns remain, particularly regarding the ON-target genotoxicity associated with protocols using nucleases. Monitoring the genotoxicity of edited cells before and after graft is essential, especially to assay potentially deleterious megabase-scale genomic rearrangements induced at the targeted locus. High sensitivity requires single-cell resolution. Here, we developed an integrated approach combining targeted single-cell DNA sequencing focused on single nucleotide polymorphism (scSNP-DNAseq) with complementary micronuclei and LOH cytometry-reporter assays. This multiplexed strategy enables orthogonal readouts to accurately monitor CRISPR-mediated genotoxicity in primary cells. Using this approach, we detected, mapped and characterized various types of induced-losses of heterozygosity (terminal, interstitial, copy-loss and copy-neutral). Our compelling workflow assessed editing-associated chromosomal instability linked to double strand break after editing. Importantly, palbociclib prevented the appearance of such genomic rearrangements in hematopoietic stem/progenitor cells without impairing cell fate or graft capability. Conversely, short-term risk was significantly increased with DNA-PKcs inhibitor AZD7648 (HDR booster) in HSPCs and fibroblasts. Fortunately, targeting HBG1/2 in Chr11p in HSPCs, scSNP-DNA-seq revealed that ON-target genotoxic events were no longer detectable after long-term xenografts, even in AZD7648-treated cells. This work demonstrates that scSNP-DNA-seq should be routinely implemented to monitor chromosomal rearrangements before and after CRISPR-edited cell infusions.

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Boutin, J., FAYET, S., MARIN, V., BERGES, C., Riandiere, M., Toutain, J., Lamrissi-Garcia, I., THIBAULT, C., Cappellen, d., Dabernat, S., Poulet, A., Francilette, M., DROUIN, N., MOREAU-GAUDRY, F., BEDEL, A.. 2025-04-10. Single-cell SNP-DNA sequencing precisely maps genotoxic events in CRISPR-edited primary cells. https://doi.org/10.1101/2025.04.04.647179

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