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

Domain: chemistry
Published: 2026-01-10
Editorial outcome: minor_revision

Abstract
Genome editing by CRISPR-Cas9 nuclease is promising for gene therapy. However, safety concerns remain. Monitoring ON-target genotoxicity is essential, especially to assay megabasic rearrangements at the targeted locus. Here, we developed a combined single-cell resolution approach with DNA sequencing focused on single nucleotide polymorphism (scSNP-DNA seq), micronuclei and LOH cytometry-reporter assays. This sensitive multiplexed strategy enables the sensitive monitoring of CRISPR-mediated genotoxicity in primary cells. Using this approach, we detect, map and characterize various types of induced-losses of heterozygosity and assessed editing-associated chromosomal instability. Importantly, palbociclib prevents the appearance of such genomic rearrangements in hematopoietic stem cells without impairing cell fate or graft capability. Conversely, short-term risk is significantly increased with DNA-PKcs inhibitor AZD7648. Fortunately, targeting HBG1/2p, scSNP-DNA-seq reveal that ON-target genotoxic events are no longer detectable after long-term xenografts. This work demonstrates that scSNP-DNA-seq should be routinely implemented to monitor chromosomal rearrangements before and after CRISPR-edited cell infusions.

Introduction
CRISPR-Cas9 genome editing holds tremendous promise for gene therapy. It offers unprecedented potential for therapeutic applications. Numerous CRISPR-based clinical trials are ongoing and the first treatments are already approved for clinical use. Despite development of new genome editing tools, most of current clinical applications still rely on CRISPR-Cas9 nuclease, and create a targeted DNA double-strand break (DSB). However, the repair of DSB can lead to complex and unpredictable outcomes, raising concerns about genotoxic risk and potential phenotypical alterations in edited cells. Both OFF-target and ON-target genotoxicity have been reported. ON-target genotoxicity includes various types of loss-of-heterozygosities (LOH): kilobase-scale interstitial copy-loss by deletions (CL-LOH), megabase-scale telomeric CL-LOH (truncations), and copy-neutral loss-of-heterozygosity (CN-LOH), which may lead to imprinting loss and disrupt imprinted genes expression. Even larger genomic abnormalities have been observed, such as whole-chromosome loss and chromothripsis. Importantly, these genomic rearrangements are observed in vitro, a few days after editing, and have been observed in many cell types, in immortalized cell lines, adult primary cells, such as hematopoietic stem/progenitor cells (HSPCs) and CAR-T cells, and even in embryos.

p53 inhibitor and DNA-PKcs inhibitors (AZD7648) are being explored to improve homology-direct repair (HDR) efficiency. However, these DNA repair pathway modulations could exacerbate post-editing ON-target genotoxicity.

In this context, the clinical deployment of CRISPR-based therapies raises significant safety concerns that must be deciphered and addressed. The first published long-term data are encouraging. Indeed, CRISPR-induced aneuploidies in CAR-T cells seem to be mitigated by long-term culture in vitro and after graft in patients. In HSPCs, kilobase-scale genotoxicity following CRISPR targeting of BCL11A erythroid enhancers on chromosome 2 (Chr2) appears to diminish after 16-weeks post-graft in mice. However, whether extra-large (megabase-scale) ON-target genotoxicity persists long term in vivo and can be mitigated remains unknown.

Addressing these questions requires highly sensitive methods with single-cell resolution. Microfluidic single-cell-DNA sequencing (scDNA-seq) should be appropriate for analyzing the genome integrity, but its accuracy was challenged for a long time by the presence of only two genomic copies per cell. Hundreds of genomic loci can now be interrogated across thousands of cells. It is used to monitor intratumoral heterogeneity, clonal evolution, and residual disease in cancer. More recently, scDNA-seq revealed aneuploidies in human tumors.

Here, we propose a multi-assay approach to longitudinally evaluate ON-target genotoxicity induced by CRISPR-Cas9 in clinically relevant cells. We combined three orthogonal single-cell methods: (i) micronuclei (MN) detection, (ii) detection of LOH using our in-house Fluorescence-Assisted Megabase-scale Rearrangements Detection (FAMReD) assay, and (iii) scDNA-seq. Importantly, by gathering single-nucleotide polymorphism (SNP) and copy number variations (CNV) analyses, we demonstrate here that scSNP-DNA-seq is a powerful assay to measure chromosomal instability following CRISPR editing, in particular CN-LOHs with high sensitivity, in vitro and in vivo.

Our orthogonal approach offers a sensitive and robust platform for assessing genome integrity of CRISPR-edited cells and ON-target genotoxicity. It enables us to precisely detect chromosomal instability, to map outcomes in human primary cells in vitro, to monitor its persistence in vivo after engraftment and to evaluate mitigation strategies.

Results
Design of the scSNP-DNA seq library and method validation for LOH quantification
To detect ON-target genotoxicity induced by CRISPR-Cas9, specifically kilobase- and megabase-scale LOH, either due to a deletion (copy-loss, CL-LOH) or without loss of genomic material (copy-neutral, CN-LOH), we designed a custom scDNA-seq library, focusing on SNPs in Chr10/Chr11. We first identified SNPs common in the general population using the dbSNP database (NCBI, 150,000 SNPs) and filtered for those with global allele frequency more than 0.5 (15,000 SNPs), as well as those with high frequency in European and African populations (403 SNPs distributed along chr10/11). We then designed probes to obtain 403 amplicons per cell containing the identified SNPs, with higher concentration near the CRISPR cut sites. We validated selected amplicons in silico with Bcftools on GIAB Coriell (NA12878 v4.2.1). Amplicons on Chr10 served as control for CRISPR targeting of Chr11, and vice-versa. After CRISPR-Cas9-editing, each amplicon from each cell was sequenced to assess genome integrity. Loss of SNPs (genotyped in unedited control cells) was used to identify and map LOH presence in each cell.

For method validation, we created a positive control cell line with a 5Mb-terminal LOH on Chr10q (LOH+). This was achieved using our previously published FAMReD system, based on human heterozygous UROS+/− fibroblasts (hFFs). By targeting ABRAXAS2 by CRISPR nuclease, located 1Mb centromeric to the UROS locus in Chr10q, we observed a slight increase in the proportion of fluorescent cells 15 days post-editing (0.1–0.2%), suggesting the occurrence of rare LOHs events at UROS locus, leading to the loss of the functional UROS+ allele, and subsequent porphyrins accumulation. We then sorted and amplified the fluorescent cells, and analyzed them using scSNP-DNA seq with Mosaic 3.0 software. We identified SNPs located along Chr10q and selected five well-sequenced SNPs in UROS+/− hFFs before editing. scSNP-DNA seq confirmed the disappearance of SNP3/4/5 and the presence of only 1 LOH profile (LOH10qI with homozygous terminal profile) in fluo+ cells (pink). As expected, more than 99% of cells exhibited an extra-large LOH extending from the CRISPR cut-site (ABRAXAS2) to the telomere. We identified a unique LOH event, linked to the allele carrying the UROS mutation (LOH10qI), thereby validating the method at the allele level. Importantly, the non-targeted Chr11 remained fully heterozygous (yellow), with no SNP loss.

scSNP-DNA seq and global UMAP reveals frequent ON-target genotoxicity after double-cut in HBG1/2 promoters in HSPCs
To investigate CRISPR-induced ON-target genotoxicity in human HSPCs, we edited the cells with the gRNA-68, which targets the two HBG1/2 promoters, as previously reported by Sharma et al. This protocol is known to predominantly induce 5kB-deletion between the two CRISPR-induced cut sites. We confirmed editing efficiency by long-range PCR and NGS analysis (Nanopore sequencing, Supplementary Fig. 1a). In parallel, we performed scSNP-DNA seq. First, we established the initial SNP profile in the unedited HSPCs. Using principal component analysis (PCA) and UMAP, we clustered 5161 edited cells, considering all SNPs, revealing three distinct populations based on 8 components and 88 detected SNPs. The majority of the cells (orange in UMAP) were heterozygous for all sequenced SNPs (Htz = A/B, white in the heatmap, right panel). UMAP analysis revealed two equally sized minor populations (2.4% and 2.2%, red and green, respectively). In these cells, 18 SNPs located between the HBG1/2p cut sites and the Chr11p telomere were lost and became homozygous (either A/A or B/B, indicated by pink or blue, respectively), mapping an extra-large LOH (~5Mb). These abnormal cells were classified as LOH11pI and LOH11pII, respectively.

To classify these LOHs as either copy-neutral (CN-LOH) or copy-loss (CL-LOH), we analyzed amplicon read-depth using the Mission Bio Mosaic pipeline to infer CNVs. Ploidy was calculated for each amplicon and cell exhibiting LOH by normalizing read counts against the mean of read counts of the “normal/orange” cell population. We observed abnormal ploidy at the chr11p extremity in both LOH populations (LOH11pI and LOH11pII). Notably, genomic area with reduced ploidy (indicated in blue in the heatmap) corresponded to extra-large LOH events spanning from the cut site to the telomere. LOH ploidy was estimated by averaging the ploidy of amplicons within the LOH genomic region for each cell in both LOH populations (red and green). Ploidy in the LOH area was reduced: 1.28 and 1.22 for LOH11pI and LOH11pII, respectively compared to a ploidy of around 2 (normal) in the centromeric region of 11p and 11q arm, and SNP3–5, located between the HBG1/2p cut sites and the 11p telomere. This finding confirms the presence of the two distinct telomeric LOHs (LOH11pI and LOH11pII) in HSPCs 4 days after transfection. This method corroborates the global genotoxicity patterns identified via UMAP-based clustering. Yet it demonstrates increased sensitivity and highlights a higher frequency of chromosomal rearrangements (7.4 vs 4.6%).

Thanks to this increased resolution, we hypothesized that smaller, kilobase-scale LOHs may be present but undetected by UMAP analysis. We then looked for smaller interstitial LOHs in the same edited cells. To test this, we substitute SNP3 and SNP4, located 0.3 and 2.8 Mb from the CRISPR cut sites) with SNP6 and SNP7, positioned closer to the cut sites, (12 and 13.6kb respectively). Clustering based on this kilobase-resolution panel identified, in addition to previously characterized terminal LOH (n=129), two additional cell populations with new SNP profiles. Specifically, 2.2% of cells (n=38) exhibited loss of SNP6-7 while retaining distal telomeric SNP5, indicative of interstitial LOHs (iLOH11pI and iLOH11pII), equally distributed between maternal and paternal alleles. These interstitial events raised the total LOH frequency to approximately 9%. No LOH were detected on the non-targeted Chr10, supporting the specificity of CRISPR-Cas9 induced genotoxicity.

To determine whether the observed LOH were due to copy number loss (CL) or copy-neutral (CN) events, we assessed CNVs in LOH-containing cells using the kilobasic SNP panel, considering all amplicons. LOH ploidy was estimated by the average of amplicon ploidies across the LOH region for each cell in each population. Among the 1855 analyzed cells, 129 cells exhibit a terminal 11p LOH. In these cells (LOH11pI and LOH11pII), the calculated mean LOH ploidies were 1.25 and 1.18, respectively, suggesting that ~80% of these LOHs resulted from terminal deletions. In the 38 cells with interstitial LOHs (iLOH11pI and iLOH11pII), mean ploidies in the affected 43kb LOH area (from the cut site to AC104389.16), were 1.44 and 1.21, respectively, again indicating a predominance of interstitial deletions. Altogether, we demonstrated the value of high-quality SNP-focused scDNA-seq for deciphering and precisely mapping the ON-target genotoxicity of HBG1/2p targeting by CRISPR-Cas9 in primary cells. Our data revealed a high frequency of both interstitial and terminal deletions at day 4.

AZD7648 increases short-term genotoxicity in human fibroblasts
Pharmacological modulators known as “HDR boosters” have been proposed to improve the HDR rate during CRISPR-mediated genome editing. To evaluate the impact of three HDR boosters on editing efficiency and genotoxicity, we treated hFFs with XL413, an inhibitor of CDC7, and two DNA-PKcs inhibitors, NU7441 and AZD7648, during ABRAXAS2 editing with a high-fidelity (HiFi) Cas9, and a single-strand oligo-desoxyribonucleotide (ssODN) template to obtain HDR. We monitored ON-target genotoxicity by a multiplex single-cell method approach combining MN assays, cytometry-based LOH detection, and scSNP-DNA seq.

PCR and Sanger sequencing revealed that, among these tested compounds, only AZD7648 significantly increases the HDR/NHEJ ratio in hFFs. The “HDR boosters” genotoxicity was first assessed using a cytometry-based MN assay at day 4 post-editing. AZD7648 treatment markedly elevated MN formation after ABRAXAS2 editing (up to 0.7% of MN/nucleus vs 0.16% in edited hFFs without AZD7648), indicating increased genotoxic stress.

To confirm this result, we edited hFFs at HBG1/2p loci. Once again, an increase of MN was observed in the presence of AZD7648, without any change in editing efficiency as measured by nanopore sequencing. The FAMReD system, developed in our lab to sensitively detect telomeric LOH by cytometry following CRISPR-Cas9 editing, confirmed the genotoxicity of AZD7648 in hFFs and showed persistence of megabase-scale terminal LOH at day 18 (0.6% of fluorescent LOH+ cells vs 0.16% in DMSO control). Since FAMReD detects only 50% of the events, the true long-term LOH frequency is double (1.2% of edited cells treated with AZD7648). A smaller genotoxic effect was observed with Nu7441 (by both MN and FAMReD analyses). The effect of AZD7648 was exacerbated in TP53−/− hFFs (6% of cells with telomeric LOH).

To quantify, map, and characterize the nature of post-editing LOH events in the presence or absence of AZD7648, the most genotoxic HDR booster, we performed a kinetic analysis of ON-target genotoxicity using scSNP-DNA seq analysis, before and after hFFs editing at ABRAXAS2 locus, with a ssODN template, with or without AZD7648. Bioinformatic analysis first identified five informative SNPs in non-edited cells, selected with stringent quality criteria to enable megabase-scale resolution. After editing, clustering based on these five SNPs revealed four cell populations with distinct SNP profiles: one population with heterozygous SNPs along the Chr10 (“WT”), two abnormal populations with allele loss for 3 SNPs 3/4/5, from each parental chromosome (designated telomeric “LOH10qI” and “LOH10qII”), and a fourth abnormal population showing loss of all analyzed SNP (Chr10pq SNP loss).

After editing in hFFs without AZD7648, we observed, on day 2, 1.5% of cells with telomeric 10q LOH, extending from the cut site to the telomere, and rare cells with complete loss of SNPs. CNVs analysis, performed only when more than five cells were available per population, revealed reduced ploidy in both LOH10qI and LOH10qII populations (1.07 and 1.37 in LOH10qI and LOH10qII respectively), demonstrating that most LOH events were due to genomic material loss (93% and 63% of deletions (CL-LOH), in LOH10qI and LOH10qII respectively). The high rate of telomeric LOH persisted at day 7 (in 1.9% of cells), with a balanced mix of CN-LOH and CL-LOH (ploidy 1.69 et 1.59). Interestingly, in the same edited cells, the frequency of LOH dropped clearly by day 20 (0.23%).

Unexpectedly, in the presence of AZD7648, no LOH was detected on day 2 using scSNP-DNA seq. We hypothesized that DNA-PKcs inhibition during a DSB induction might slow down cell division, delaying the onset of ON-target genotoxicity. We therefore analyzed the cell cycle of hFFs exposed to AZD7648. When a DSB occurs concomitantly with AZD7648 treatment, hFFs tend to accumulate in the G0 phase at day 2. These results may explain the absence of detectable LOH immediately after editing. At day 7, however, a high rate of LOH was observed (3.9% of cells), with a mixture of CN-LOH and CL-LOH (ploidy 1.31 et 1.48 in LOH10qI and LOH10qII respectively). This genotoxicity was partially maintained at day 20 (1.37%), suggesting a delayed onset and sustained maintenance, compared to without AZD7648. At day 20, in the presence of AZD7648, the persistent LOHs were predominantly copy-neutral (ploidy 1.68 and 1.59 in LOH10qI and LOH10qII respectively). No LOH was detected on the non-targeted Chr11.

Among all tested HDR enhancers, AZD7648 reached the highest short-term genotoxicity in hFFs. Kinetic reveals that LOHs, initially resulting from DSB and chromosomal truncations, tend to convert into copy-neutral LOHs and diminish in frequency at day 20.

AZD7648 increases short-term ON-target genotoxicity in HSPCs targeting UROS (Chr10q)
Because DNA repair pathways are cell-type dependent, we next investigated the genotoxicity of HDR boosters in HSPCs. We targeted the UROS locus in Chr10q in the presence or absence of HDR boosters. PCR and Sanger sequencing confirmed an increase in the HDR rate upon AZD7648 treatment (77% vs 12%). Again, AZD7648 exposure during editing led to a dramatic increase in MN formation (up to 3–4% of MN/nucleus), suggesting that modulating DNA repair pathways during DSB repair also elevates genotoxicity in HSPCs. DNA staining by DAPI and fluorescence microscopy of edited HPSCs further confirmed the increase in nuclear abnormalities upon AZD7648 treatment.

To monitor the kinetics of unwanted genome editing outcomes in HSPCs, we designed a time-course scSNP-DNA seq analysis with or without AZD7648. HSPCs were edited, cultured, and collected on days 4, 7, and 14 for scSNP-DNA seq. We identified four cell populations post-editing: “WT” without LOH (all SNP heterozygous), telomeric LOHs “LOH10qI” and “LOH10qII” (SNPs 3/4/5 loss), and whole-chromosome LOH “LOH10pq” (all SNPs lost). Without AZD7648, CRISPR editing led to 2–4% of rearranged cells, mostly showing telomeric LOHs (“LOH10qI and LOH10qII”) from day 4 to day 14, confirming that even a single DSB can induce ON-target genotoxicity. AZD7648 exposure significantly increased the proportion of cells with telomeric LOHs throughout the time-course (day 4 to day 14), reaching ~7%, a threefold increase compared to CRISPR alone. These results indicate that telomeric LOHs are stable in vitro over a two-week period. No LOH was detected on the non-targeted Chr11, even with AZD7648. Under all conditions, with or without AZD7648, and whatever the time point, the majority of LOHs were copy-neutral, with ploidy values around 2 (ranging from 1.74 to 2).

Taken together, these data demonstrate that AZD7648 induces both short- and medium-term ON-target genotoxicity in HSPCs, with immediate cellular stress (MN and nuclear abnormality formations) and the persistence of extra-large copy-neutral LOH events in vitro for up to two weeks.

Palbociclib prevents post-editing ON-target genotoxicity in HSPCs
Mitigating ON-target genotoxicity is essential for the safe development of CRISPR-based gene therapy using nucleases. DSB-induced large-scale genotoxicity is thought to mainly occur in dividing cells. We hypothesized that G0/G1 cell cycle arrest could mitigate chromosomal rearrangements observed in HSPCs. In a previous work, we demonstrated that palbociclib, a CDK4/6 inhibitor, effectively blocks fibroblasts in the G0/G1 phases. We therefore investigated whether palbociclib could transiently arrest HSPCs in G0/G1 during editing and thereby prevent genotoxicity in clinically relevant human HSPCs.

We first monitored its impact on (i) HSPCs cell cycle progression, (ii) editing efficiency (via long-range PCR and nanopore sequencing), and (iii) genotoxicity using multiplexing single-cell approaches (MN and scSNP-DNA seq). We optimized the dose and timing of palbociclib to transiently block HSPCs in the G0/G1 phases. Just after thawing, nearly all HSPCs were in G0/G1 phases (99%). While this proportion decreased after 12h in culture, palbociclib maintained a higher fraction of cells in G0/G1 (84% vs 63% without palbociclib). A 36h-palbociclib exposure further enhanced this effect (92% of cells in G0/G1 phase vs 70% without palbociclib), which was reversed upon drug removal (81% G0/G1 cells with palbociclib after 36h-treatment and a 48h-clearance/washout vs. 76% in untreated cells). For comparison, cytokine starvation was also tested to reduce cell proliferation. Comparison between palbociclib vs cytokine starvation revealed that palbociclib was more efficient than 3ng/mL cytokine starvation to synchronize the cells. Complete cytokine starvation (0ng/mL) is equally potent to palbociclib to arrest the cells in G0/G1 and block proliferation but more toxic. Thus, palbociclib was selected for transient HSPCs cell cycle arrest in the remainder of the study.

We then edited HBG1/2p in HSPCs in the presence of palbociclib. Treatment during editing maintained >95% of cells in G0/G1 phases without compromising editing efficiency.

To evaluate short-term ON-target genotoxicity, we first analyzed nuclear abnormalities in HSPCs at day 2 by DAPI staining (MN, nuclear buds…). Fluorescence microscopy revealed that CRISPR targeting of HBG1/2p induced a high rate of abnormal nucleus, hallmarks of genomic instability (around 20% of cells with abnormal nucleus). Palbociclib reduced this frequency by more than twofold.

To confirm, we performed MN analysis by cytometry. HBG1/2p targeting by CRISPR led to a high MN rate, which was again reduced over twofold with palbociclib in HSPCs (from 1.8% to 0.9%).

To track chromosomal rearrangements, we collected cells on day 4 to perform scSNP-DNA seq using the kilobasic panel. Total LOH frequency (telomeric + kilobasic) dropped from 9.2% in edited controls to 2.9% or 2.1%, with 24h or 48h of palbociclib exposure, respectively. These data demonstrate that 24-h exposure to palbociclib is sufficient to protect HSPCs from ON-target genotoxicity. In the remaining rearranged cells, all four LOH subtypes were still present, but palbociclib altered their distribution. Specifically, telomeric LOH frequency dropped from 6.95% to 1% after 48h of palbociclib treatment, a sevenfold reduction, while interstitial LOH frequency was only reduced twofold (from 2.25% to 1.1%). Ploidy analysis of each LOH event showed that the proportion of CL-LOH and CN-LOH was unchanged after 24h of palbociclib (ploidy ~1.3, corresponding to 70% deletions in both conditions). Altogether, these findings demonstrate that transient G0/G1 arrest by palbociclib (12h pre-treatment + 24h post-editing exposure) effectively protects HSPCs from DSB-induced damages (preferentially megabase-scale LOH), without compromising editing efficiency via NHEJ.

We hypothesized that G0/G1 maintenance by palbociclib may prevent genotoxicity by promoting end-joining. Because MMEJ is absent in G0/G1, we challenged NHEJ implication in palbociclib preventing effect. To test this, we used the highly sensitive FAMReD system to evaluate the effect of concomitant NHEJ inhibition by AZD7648 during palbociclib exposure. Interestingly, in the presence of AZD7848 plus palbociclib, megabasic LOH rate was exacerbated. However, cell cycle analysis showed that cells remained synchronized in G0/G1. These data demonstrate that the protective effect of palbociclib relies on NHEJ activity to prevent megabase-scale chromosomal rearrangements. Sequencing data further support the NHEJ importance in palbociclib-treated cells with an increase of small indels proportion.

Palbociclib improves cell graft capability and hematopoietic reconstitution of HSPCs
By inducing G0/G1 cell cycle arrest in HSPCs, palbociclib could theoretically impair cell proliferation and impact in vitro/in vivo hematopoietic function. We first carried out an in vitro test of proliferation. As expected, palbociclib transiently reduced HSPCs proliferation in hematopoietic liquid culture, similar to complete cytokine starvation (0ng/mL) but with less toxicity.

This proliferation slow-down could affect in vivo HSPCs properties. To assess longer-term consequences, we grafted equivalent numbers of HSPCs pre-treated or not with palbociclib into NOD-scid IL2Rgamma null (NSG) mice. After 17 weeks post-transplantation, bone marrow (BM) chimerism (% hCD45+ cells) was higher in mice receiving palbociclib-treated cells. Hematopoietic lineage reconstitution (hCD33+ and hCD19+ cells) in BM was comparable between conditions, demonstrating that palbociclib does not impair multilineage differentiation.

To quantify grafting capacity, we performed limiting dilution analysis (LDA) across 120 mice. LDA confirmed that palbociclib does not compromise HSPCs engraftment and even enhances it. Indeed, the number of transplanted mice is greater with palbociclib than without. For example, among mice transplanted with 100 and 500 cells, only those incubated with palbociclib show successful engraftment (2/20 vs 0/20 mice). This result was confirmed in secondary recipient mice, with a higher number of engrafted mice in the palbociclib group (4/10 vs 2/10). The frequency of SCID Repopulating Cells (SRCs) was 2.6-fold higher with palbociclib (1/1360, CI [1/740-1/2500]), compared to untreated controls (1/3541, CI [1/1700-1/7350]).

These findings suggest that, by forcing HSPCs to be in the G0/G1 phases, palbociclib could modulate their stemness properties. Phenotypic analysis after 36h of exposure showed an increased frequency of stem cell population (CD34+/CD38low/CD90+/CD133+) (from 12% to 26%), suggesting the maintenance or selection of stem cells, or possible progenitor dedifferentiation. This is supported by the upregulation of hematopoietic stemness-associated genes such as CXCR4, TAL1, and RUNX1 in HSPCs treated with palbociclib for 36h.

To confirm the palbociclib’s positive effect on engraftment in context of nuclease-based genome editing, we targeted ABRAXAS2 locus in HSPCs, with or without palbociclib exposure (12h pre-treatment + 24h post-editing). Editing efficiencies were very high in both conditions. Following palbociclib removal, equal numbers of cells were transplanted into NSG mice. Again, chimerism was higher in the palbociclib group, without altering CD19+ and CD33+ lineage distribution. Together, these results demonstrate that palbociclib is not deleterious, and in fact enhances HSPCs engraftment. Its use during CRISPR protocols could therefore be explored as a strategy to prevent ON-target genotoxicity while preserving or enhancing edited-HSPCs transplantation.

Chromosomal rearrangements disappear after long-term engraftment
To evaluate longer-term impact of LOH events on HSPCs proliferation and function, we engrafted CRISPR-edited HSPCs (targeting in HBG1/2p) into NSG mice for in vivo genotoxicity monitoring. Prior graft, we confirmed editing efficiency. In parallel, we observed the immediate genotoxicity by MN assay.

We grafted 400,000 edited human HSPCs per mouse, 24h after CRISPR editing, with or without AZD7648 exposure (n=2 mice per condition). We then analyzed LOH frequency by scSNP-DNA seq in “before graft” and 3 months after graft (in vivo) to assess persistence of genotoxicity in BM HSPCs. For that, we harvested BM cells from mice 90 days after graft. We obtained high chimerism (hCD45+ cells) highlighting a good hematopoietic reconstitution. Human hematopoietic CD45+ cells were sorted by magnetic beads and purity confirmed before scSNP-DNA seq analysis. Before graft, 3.6% and 10.5% of cells exhibited megabase-scale telomeric LOH (from HBG1/2p to the 11p telomere) in the absence or presence of AZD7648, respectively. However, at day 90 post-engraftment, no genotoxicity was detected, even though edited alleles persisted. These findings demonstrate the complete disappearance of rearranged cells over time in vivo, including in the more genotoxic AZD7648 condition.

To sum up, these data establish that scDNA seq can be used to monitor in vivo ON-target genotoxicity and show that Chr11p LOH+ cells do not persist in vivo, even after editing with AZD7648.

Methods
Cell culture
Human foreskin fibroblasts immortalized with hTERT (hFFs) were from ATCC® (CRL 4001, BJ-5ta). They were partially inactivated for UROS using a ribonucleoprotein (RNP) made of Cas9 protein complexed with a gRNA targeting UROS exon 4. hFFs were maintained in DMEM, high-glucose (4.5g.L-1), L-Glutamine (1g.L-1) and pyruvate (Gibco® by Life-technologiesTM, Carlsabad, CA, USA, catalog n°31966047) supplemented with 10% fetal bovine serum (FBS) (Eurobio, Les Ulis, France, Catalog #CVFSVF00-01), 100U/mL penicillin and 100μg/mL streptomycin (Gibco® by Life-technologiesTM, Carlsabad, CA, USA; catalog #15070063), 10µg/mL ciprofloxacin (Biogaran™, Colombes, France) and 0.5µg/mL amphotericin B (Sigma-Aldrich®, Saint Louis, MO, USA, catalog #A2942).

Human CD34+ HSPCs were isolated from the cord blood. Briefly, mononuclear cells were isolated by Ficoll gradients. HSPCs were purified according to the manufacturer’s instructions (hCD34-Positive Selection kit II, from Stem Cell Technologies, Vancouver, BC, Canada, catalog #17865), and purity was analyzed by flow cytometry using phycoerythrin (PE)-conjugated anti-CD34 antibody (clone 561, Biolegend, San Diego, CA, USA). The CD34+ cell purity was >90%. Cryopreserved HSPCs were thawed and cultured in expansion medium consisting in StemSpan SFEM (Stem Cell Technologies, catalog #09600) supplemented with Flt3-L (100ng/mL, catalog #GMP300-19-50UG), SCF (100ng/mL, catalog #GMP300-07-50UG), hTPO (100ng/mL, catalog #GMP300-18-50UG), (all from Peprotech, Cranbury, NJ, USA), 100U/mL penicillin, and 100μg/mL streptomycin (Gibco® by Life-technologiesTM, Carlsabad, CA, USA, catalog n°15070063), 10µg/mL ciprofloxacin (Biogaran™, Colombes, France) and 0.5µg/mL amphotericin B (Sigma-Aldrich®, Saint Louis, MO, USA, catalog n°A2942).

All cells were grown at 37°C with 5% CO2 levels. All cell lines were tested for mycoplasma.

To increase HDR, we added 3 “HDR boosters” in the medium for editing: NU7441 (2.5µM, 18h before transfection and for 2 days after transfection, from Selleckchem, Houston, US, catalog #S2638), AZD7648 (1µM, 18h before transfection and for 2 days after transfection, from MedChemExpress, Monmouth Junction, New Jersey, US, catalog #HY-111783) or XL413 (20µM, for 2 days after transfection, no cell exposure performed before transfection, from Selleckchem, Houston, US, catalog #S7547). Dimethyl sulfoxide (DMSO, from Sigma-Aldrich®, Saint Louis, MO, USA, catalog #D2438), used to dissolve these compounds was used as a negative control. To prevent genotoxicity, cells were synchronized in G0/G1 phase by incubation with palbociclib (1µM, Sigma Aldrich, Saint Louis, MO, USA, catalog #PZ0383) 18h before and 24h or 48h (for scDNA-seq) after RNP transfection.

Editing tools
The crRNA for the ABRAXAS2 and UROS targets (Ch10q) was designed using CHOP-CHOP software (chopchop.cbu.uib.no) (ABRAXAS2 5′-CCATCGAGTGGGAGGATCT-3′ and UROS exon 4: 5′-GGAAGCAGCAGAGTTATGTT-3′, Supplementary Table 1). The crRNA chosen for editing of the HBG1 and HBG2 promoters (Chr11p) is an already published gRNA (gRNA-68). The different components of the CRISPR-Cas9 system were combined to form a RNP (all ordered at IDT, Integrated DNA Technologies, Coralville, IA, USA). For this, 17µg of Alt-R® S.p. HiFi Cas9 Nuclease V3 when a ssODN was added (abbreviated hereafter as HiFiV3Cas9, Integrated DNA Technologies, Coralville, IA, USA, catalog #1081061) or Alt-R™ S.p. Cas9 Nuclease V3 (abbreviated hereafter as V3Cas9, Integrated DNA Technologies, Coralville, IA, USA, catalog #1081059) and 6.5µM of sgRNA, were mixed and then incubated for 10–20 min at room temperature before electroporation. For HDR booster experiments, 6.5µM of ssODN template was added (ABRAXAS2 5′-TCTGCTTCTTCATCCTAGTTCACTCACTTTGCCCCCAGCTGCCTCCATCGAGTGGGAGGAAGAGCTCGACAGAGCCTGTTCACTCTAGTCATTTATGCAACAGACATCAATGGAAACAT-3′, and UROS 5′-CTGAAGATTACGGGGGACTCATTTTTACCAGCCCCAGAGCAGTGGAAGCAGCAGAGCTCTGTTTAGAGCAAAACAATAAAACTGAAGGTGAGGGTGGGTCTGCTGTGGATTCCACTGGAC-3′, Supplementary Table 2). Finally, 3.9μM of Alt-R Cas9 Electroporation Enhancer solution (Integrated DNA Technologies, Coralville, IA, USA, catalog #1075916) was added to the RNP complex to improve electroporation efficiency.

Cells were transfected by electroporation using the Nucleofector 4D AMAXA electroporation system (Lonza®, Bale, Switzerland). In brief, 2.105 to 3.105 depending on the cell type (HSPCs and hFFs) were resuspended in P3 Primary Cell Line 4D-Nucleofector® (Lonza, Bale, Switzerland, catalog #V4XP-3024) and added to the RNP complex. Then cells were nucleofected using DO-100 and CZ-167 programs respectively.

Editing efficiency
Genomic DNA of edited cells, and their associated controls, was extracted using Nucleospin® Tissue (Macherey-Nagel®, Düren, Germany, catalog #740952.250) according to the manufacturer’s protocol. The genomic region flanking the expected cut-site was amplified by PCR (HotStarTaq Plus DNA polymerase, Qiagen®, Venlo, Netherlands, catalog #203605) with adequate primers (ABRAXAS2 targeting F: 5′-CCTGGCTCACTCACTCTGTG-3′, R: 5′-AGGCCAGTTCAAACGCTCTT-3′; UROS targeting F: 5′-TAGTTCCAGGCACATAGTAAGCAC-3′, R: 5′-TCCCAAGGCAGAGTCTGTGA-3′; HBG1/2 promoters targeting F: 5′-GCTTTGTTGCGCAGGTCAACATGTAT-3′, R: 5′-GGGGCTGCTCTGCCTATGCAGTAGTC-3′), Supplementary Table 3. PCR products were purified with Nucleospin® Gel and PCR Clean-up (Macherey-Nagel®, Düren, Germany, catalog #740609.50S). Sanger sequencing was done on purified PCR products and sequenced by LIGHTRUN (GATC Biotech, Konstanz, Germany). Sanger sequencing data were analyzed using ICE v2 CRISPR Analysis tool (ICE) software (Synthego, Redwood City, CA, USA) and DecodR v356. Purified PCR products from unedited cells were used as control chromatograms.

To validate CRISPR-Cas9 efficiency at HBG1/2p in HSPCs, we used Nanopore NGS. Briefly, 7 days after editing, genomic DNA was extracted from cell pellets using Nucleospin® Tissue (Macherey-Nagel®, Düren, Germany, catalog #740952.250) according to the manufacturer’s protocol. The genomic region flanking the edited site was amplified by Long Range PCR (LA Taq, Takara Bio, Kusatsu, Japan), with adequate primers. Sequencing library was prepared using Native Barcoding Kit 96 V14 (Oxford Nanopore Technologies, Oxford, United Kingdom, catalog #SQK-NBD114.96) according to the manufacturer’s protocol. Sequencing was then performed using a MinION Mk1B device and a Flow Cell R10.4.1 (Oxford Nanopore Technologies, Oxford, United Kingdom, catalog #FLO-MIN114) to reach at least 3000 qualify-reads per sample (Q score > 9). After sequencing, super-accurate base calling was performed using MinKNOW (v24.02.6) and fastq were aligned on human reference genome GrCH38 (GCA_0000001405.15) using a home-made Bash (v5.1.16) script based on Epi2Me (v5.1.9) wf-alignment pipeline (v1.1.2). Then, BAM were analyzed using a homemade bioinformatic pipeline in Python (v3.13.2) using pysam (0.22.0). Briefly, it classifies reads into categories (WT, small inDels, 5kb deletions, truncated etc…) based on CIGAR string interpretation. Results are summarized in .csv tables using pandas (v2.3.1). All scripts and documentation are available at: https://github.com/BioGO-BRIC/HBG1-2---ONT-analysis.

Micronuclei analysis by cytometry
The MicroFlow In Vitro-250/50 Kit (Litron Laboratories, Rochester, NY, USA, catalog #INVITRO250/50) was used for all MN assays, following the manufacturer’s instructions. Briefly, cells were stained and lysed following the protocol described in the kit. Following harvest, a photo-activated dye Dye 1, Ethidium monoazide, was used to stain apoptotic and necrotic cells then the cells were washed. Subsequently, healthy chromatin was stained with the nucleic acid Dye 2, SYTOX Green. Cells could be stored up to 24h at ambient temperature, protected from light, or refrigerated for up to 72h, prior to analysis. Using a BD Accuri C6 Plus (BD Biosciences, Franklin Lakes, NJ, USA), flow cytometric analysis distinguished 10.000 to 20.000 real MN (SYTOX Green only) from nuclei, based on their weaker DNA-associated fluorescence signal. At least 10,000 cells per condition are analyzed.

MN analysis by nuclear fluorescent imaging
Cytogenetic examination was carried out on interphase nuclei on HSPCs after fixation by a solution of 3:1 methanol/glacial acetic acid fixative. DAPI (Sigma Aldrich, Saint Louis, MO, USA, catalog #MBD0015) staining was performed onto a microscopic slide and MN examination was evaluated under a fluorescent microscope Leica DM5500 B (Leica microsystem, Wetzlar, Germany) with the 63× objective lens (i.e., 630× magnification). The number of cells visualized for each condition is indicated in the legend of each figure. Blind count of MN with a medical cytogeneticist (J.T. in author list).

LOH analysis by FAMReD system (cytometry)
FAMReD is a cytometry-based system to detect induced LOHs in Chr10q with or without TP53 inactivation. On day 15 post-editing of hFFs UROS+/−, 0.3 mM of 5-ALA (Sigma Aldrich, Saint Louis, MO, USA, catalog #A7793) were added to culture media. After 16h of exposure (overnight), cells were washed twice with DPBS 1× (Gibco, by Life Technologies, Carlsbad, CA, USA, catalog #14190144) and put back in fresh media. Upon LOH, loss of UROS can be detected by the appearance of fluorescence due to porphyrin accumulation. Following 8h of clearance, fluorescent cells were quantified by flow cytometry using a BD Accuri C6 Plus (BD Biosciences, Franklin Lakes, NJ, USA). UV-sensitive porphyrins were excited at 488nm and the emitted wavelength was approximately 667nm, detected by the PE-Cy5A PMT channel. FL-1 is a control green-fluorescent channel used to exclude auto-fluorescent cells. The system only detects 50% of the events when the cells become UROS−/−. The true LOH rate is double.

Cell cycle
To evaluate impact of palbociclib and HDR boosters on hFFs and HSPCs cell cycle, cells were harvested and washed with DPBS (Gibco® by Life-technologiesTM, Carlsbad, CA, USA, catalog #14190144) with 2% FBS (Eurobio, Les Ulis, France, catalog #CVFSVF00-01) and EDTA 2mM (Éthylènediamine-tétraacétate disodium dihydrate, Euromedex, Souffelweyersheim, France, catalog #EU0007). Cells were fixed and permeabilized using the True-Nuclear Transcription Factor Buffer Set following the manufacturer’s instructions. True-nuclear Fixation buffer (1×, from True-Nuclear Transcription Factor Buffer Set, 424401, BioLegend, San Diego, California, USA, catalog #424401) was added and incubated for 45min in the dark at ambient temperature. Subsequently, a true-nuclear 1× Perm Buffer (from the same set, BioLegend, San Diego, California, USA, catalog #424401) was used twice to wash. Cells were then stained with a PE mouse anti-Ki67 antibody (BD Biosciences, Franklin Lakes, NJ, USA, catalog #556027) for 30min in the dark at ambient temperature and washed with true-nuclear 1× Perm Buffer (from the same set, BioLegend, San Diego, California, USA, catalog n°424401). Pellet was harvested in DPBS with FBS 2% and EDTA 2mM and resuspended in a 2µg/mL Hoechst solution (bisBenzimide H 33342 trihydrochloride, Sigma-Aldrich®, Saint Louis, MO, USA, catalog B2261). Samples were stored at +4°C overnight before analysis performed on a BD LSR Fortessa™ Cell Analyzer (BD Biosciences, Franklin Lakes, NJ, USA). Data were analyzed with BD FACSDiva™ Software (BD Biosciences, Franklin Lakes, NJ, USA).

Design of custom Tapestri panel for sequencing
The panel comprises 403 probes mainly across the 10 & 11 human autosomes (Supplementary Table 1). To identify candidate target regions for the panel, we used the dbSNP (NCBI dbSNP Build 155, hg19) and considered only variant SNPs with a major allele frequency between >0.5 and <0.7 without discrepancy on certain ancestry group (e.g., global MAF=0.5 and <0.5 in African and Caucasian ancestry in GnomAd v4). In Silico validation of the SNP design was assessed by testing it on the CORIELL (GIAB consortium, sample NA12878, HG001) using bcftools (v1.18). The objective of >30% informative SNPs was largely achieved with 63%. All candidate SNPs were submitted to the Tapestri Panel Designer to generate a panel design and ensured that the designed probes targeted the candidate SNPs and had similar GC contents. Support for the custom panel design and synthesis of the panel was provided by Mission Bio using v3 Tapestri Chemistry (Mission Bio, South San Francisco, CA, USA).

Single-cell DNA-seq
Single-cell DNA-seq was performed using the Tapestri platform (Mission Bio, South San Francisco, CA, USA) according to the manufacturer’s specifications with kit v3. Briefly, cryopreserved human cells were gently thawed, washed, and quantified using a Countess II cell counter (Thermo Fisher Scientific, Waltham, MA, USA). The cells were then diluted to a concentration of 2000 to 3000 cells per μL in the cell buffer. Next, 35μL of cell suspension was loaded onto a microfluidics cartridge and cells were encapsulated on the Tapestri instrument, followed by cell lysis with protease digestion, followed by heat inactivation using a thermal cycler. The cell lysate was reintroduced into the cartridge and processed such that each cell possessed a unique molecular barcode. Amplification of the custom-targeted DNA regions was carried out by incubating the barcoded DNA emulsions in a thermocycler following Mission Bio’s specifications. Emulsions were broken, DNA digested and purified with AMPure XP beads (Beckman Coulter, Brea, CA, USA, catalog #A63881). The beads were pelleted and washed and DNA libraries were generated. Final libraries were purified with AMPure XP beads. All libraries were sized and quantified using an Agilent Fragment Analyzer and pooled for sequencing on an Illumina NovaSeq 6000 S1 flow cell with 2×150 bp at Gustave Roussy. A summary of metrics (mean reads/cell/amplicon, reads mapped to target, dropout rate, and amplicon >10×) are provided in Supplementary Table 4. Raw data for each run and sample are available in data availability section (Zenodo and NCBI SRA database).

Tapestri single cell DNA analysis
Single-cell DNA sequencing data were obtained from primary cells and analyzed using the Tapestri Mosaic pipeline and customized versions of Mission Bio’s available Jupyter notebook (Mission Bio, South San Francisco, CA, USA). Quality control steps included the removal of low-quality or missing genotype cells as per the pipeline’s automated filters. Variants were annotated using the default settings provided by the manufacturer in the Tapestri Mosaic suit.

For HSPCs edited in HBG1/2 promoters, clustering method using PCA and UMAP, based on 88 heterozygous SNPs on chromosome 11 (Chr11), was performed. To look for genome rearrangements, we performed a dimensionality reduction using PCA and Uniform manifold approximation and projection (UMAP) for non-linear embedding. Clustering was performed using hierarchical density-based spatial clustering of applications with noise (HDBSCAN) with a minimum cluster size of 22, enabling the identification of distinct genomic subpopulations. The optimal number of principal components (PCs) was determined through an elbow plot analysis, identifying the inflection point where the explained variance ratio begins to plateau. This transition, occurring around 8 PCs (determined via visual inspection), represents the balance between maximizing data variance retention and minimizing noise contribution. UMAP were then generated using the selected PCs as input, employing the Euclidean distance metric with default Tapestri Mosaic parameters. Clustering was carried out in an unsupervised manner using the hdbscan algorithm.

In case of selection of 5 high-quality SNPs, variant annotation manufacturer settings were not modified. LOHs analysis was performed by adjusting the max ADO score at 0.2 and the Minimum clone size at 0.01. Only heterozygous SNPs (0.40 < AF < 0.60) present in at least 60% of the cells were retained.

SNP profiles to cluster cells were retained if they contained at least 6 cells. The CNVs analysis was only performed in the LOH subtypes with at least 6 cells. The filters were set up to 50 for amplicon completeness, 10 for amplicon read depth, and 10 for mean cell read depth. CNVs were inferred from single-cell amplicon read-depth data using the Mission Bio Mosaic pipeline. Ploidy was calculated for each cell and each amplicon by normalizing read counts against the mean of the “normal” cell population, which was designated as the diploid reference (considering ploidy 2 for normal cells, without LOH). LOH ploidy was calculated by averaging the ploidies obtained for amplicons encompassing the LOH. scDNAseq Global Metrics are available in Supplementary Table 4.

Proliferation, cell death, in vitro phenotype and stemness gene expression in HSPCs. Proliferation was assessed with sequential cell numeration following or not 36-h exposure to Palbociclib. Cell numerations were performed using KOVA slides 10 counting chambers (Alltrista Plastics Ltd., USA; distributed by Labellians, France, Ref. 87144F) according to the manufacturer’s instructions.

Apoptosis was detected by staining the cells with anti-human APC Annexin V (#640920 Biolegend, San Diego, CA, USA) and propidium Iodide (#596552, Dutscher) solution followed by flow cytometry analysis. Cells that were propidium iodide (PI) negative and Annexin V negative were considered healthy, cells, PI negative and Annexin V positive cells were considered apoptotic, and cells that were positive to both PI and Annexin V considered necrotic.

To evaluate phenotype (stemness) of HSCPs, cells were stained with human FITC anti-CD38, and human PeCy7C anti-CD90 antibodies, from BD Biosciences (respectively #567148 and #561558, BD Biosciences, Franklin Lakes, NJ, USA) human PE anti-CD34 and human APC anti-CD133 from Biolegend (respectively #343506 and #372806, Biolegend, San Diego, CA, USA), and analyzed by cytometry using FACS Canto II from BD.

Stemness Hematopoietic gene expression was evaluated by RTqPCR. Total RNAs were extracted using NucleoSpin RNA Plus XS Microkit (Macherey-Nagel®, Düren, Germany). mRNAs were reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Life Technologies Corporation, Carlsbad, California, USA). In all cases, qPCR was performed using GoTaq® qPCR Master Mix (Promega, Fitchburg, Wisconsin, USA) in a CFX96TM Real-Time System (Bio-Rad Laboratories, Hercules, California, USA). Primers were synthesized by Eurogentec (Liège, Belgium). Previously published primers were used.

Hematopoietic engraftment in immunodeficient mice
NSG mice were obtained from the Jackson Laboratory and bred in-house (animal facilities, University of Bordeaux). Female mice between 8 and 10 weeks old were used for all experiments. The accommodation conditions are maintained at a temperature of 21°C±1.5°C, with standard 12-h cycles and humidity levels between 40 and 65%. Prior to cell injection, mice were conditioned by two intra-peritoneal injections of 20 mg/kg busulfan (Busulfan; Fresenius Kabi), at 24 h intervals. Two days after the second injection of busulfan, unmanipulated and treated hCD34+ cells were IV-injected into recipient mice under anesthesia (Isoflurane, 5% at initiation then 2.5%). Depending on our objectives, mice were sacrificed at different time points (weeks) after transplantation, and BM was harvested from femurs. Total BM cells were labelled with BV421 anti-murine TER119 (BD, 740686), BV421 anti-murine CD71 (BD, 740667), BV421 anti-murine CD45 (BD, 563890) to exclude murine cells from the analysis. FITC anti-human CD45 (Beckman Coulter, A07782), PE anti-human CD19 (Beckman Coulter, A07782), and APC anti-human CD33 (Beckman Coulter, A07782) antibodies. Human vs. mouse chimerism was analyzed by flow cytometry with FACS Canto II.

Palbociclib effect
For limiting dilution assay (LDA) with or without palbociclib, ten mice per group (cell dose) received 25, 50, 100, 500, or 3000 CD34+ cells non-treated or treated with palbociclib. Mice were sacrificed 17 weeks after transplantation and their BM analyzed by flow cytometry as mentioned above. SRC frequencies with 95% IC were determined using the extreme limiting dilution analysis webtool accessible at http://bioinf.wehi.edu.au/software/elda/. For secondary transplantation, to evaluate a potential effect of palbociclib on long-term SRC, 3000 non-treated or treated CD34+ cells were injected in primary mice (10 mice per group). Eight weeks after transplantation, mice were sacrificed, and BM was harvested from femurs. The total cells from one femur of each primary mouse was suspended in 20µL of StemSpan SFEM II medium (Stem Cell Technologies) and then injected directly in the BM of one respective secondary recipient by intra-bone injection. Mice were sacrificed 17 weeks after transplantation and their BM analyzed by flow cytometry as mentioned above.

For Fig. 6c, in order to test palbociclib function on engraftment, 5 mice per group received 10,000 CD34+ cells treated or not with palbociclib (n=2 independent experiments). For Fig. 6h, 5 mice per group received 50000 cells, edited or not in ABRAXAS2 by CRISPR-Cas9, with or without palbociclib (n=2 independent experiments). Mice were sacrificed 17 weeks after transplantation and their BM analyzed by flow cytometry as mentioned above.

Long-term in vivo genotoxicity monitoring (with or without AZD7648)
To assess the potential genotoxicity of HBG1/2p-CRISPR editing on CD34+ cells, 400,000 CD34+ edited without or with AZD7648 were injected intravenously into recipient mice (2 mice per group). 90 days later, the mice were sacrificed, and the whole BM cells from femurs and tibias were harvested prior to human CD45+ cell selection by magnetic beads (Miltenyi Biotech, hCD45 microbeads) and scSNP-DNA seq experiment.

Statistical analysis and reproducibility
When possible, experiments were conducted at least 3 times independently. No statistical method was used to predetermine sample size. No data were excluded from the analyses. The experiments were not randomized. The Investigators were not blinded to allocation during experiments and outcome assessment. Only DAPI analysis of MN was performed blindly. Statistical significance was inferred when necessary. Exact distinct and independent experiments size is indicated in each legend (n). GraphPad Prism 10 software (GraphPad, Boston, MA, USA) was used for statistical analysis. Results are presented as mean ± SD, frequencies, or mean ± SEM (indicated in each figure legend). The parametric T test (two-sided) was used when distribution was Gaussian/normal (Shapiro–Wilk test). The non-parametric Mann–Whitney test (one-sided) was used to compare two groups when distribution was not normal. One-way ANOVA (two-sided), complemented with the unprotected Fisher’s Least Significant Difference test, was used to compare more than two groups. Percentages of LOHs in HSPCs were compared by the Chi-square test.

Ethics statement
Our research complies with all relevant ethical regulations. Cord blood Human CD34+ HSPCs were obtained in collaboration with EFS Nouvelle Aquitaine-Limousin (24SPL02 agreement for the cessation of between EFS and Bordeaux University), according to the hospital’s ethical institutional review board and with the mother’s informed consent. Prior to donating peripheral blood, each donor read an informative document and completed a questionnaire allowing the possibility to be opposed to the use of discarded cells for research.

The animal experiments presented in this paper were authorized by the Ethical Committee of Gironde (CEEA50, 2022/06/03), France (ref. APAFIS #35068-2022013110253304 v8). All animal experiments were performed in agreement with French regulation—licenses granted to C.D. and P.B.G. through numbers. 3307010 and A3312054, respectively.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Summary\nThis manuscript presents a targeted single-cell DNA sequencing (scDNA-seq) method using a custom SNP panel to map\ngenotoxic events in CRISPR-edited primary human cells. The authors apply this approach to human fibroblasts and CD34⁺\nhematopoietic stem/progenitor cells (HSPCs), targeting loci on chromosomes 10 and 11. They demonstrate that CRISPR-\ninduced double-strand breaks (DSBs) can lead to substantial genomic damage, including large deletions and loss of\nheterozygosity (LOH), even at on-target sites. Importantly, they show that the DNA-PKcs inhibitor AZD7648, used to promote\nhomology-directed repair, significantly increases the frequency of terminal and interstitial LOH. Conversely, transient cell\ncycle arrest with palbociclib (CDK4/6 inhibitor) reduces these genotoxic outcomes by 3- to 5-fold. The study combines\ntargeted scDNA-seq with orthogonal readouts such as micronuclei formation and LOH reporter assays, providing a\ncompelling workflow for assessing editing-associated chromosomal instability.\nMajor Comments\n1) Novelty\nWhile the study is methodologically strong, the novelty of the findings is not clearly delineated. Several key observations—\nsuch as CRISPR-induced LOH, the exacerbating effect of DNA-PKcs inhibition, and the protective role of G1 arrest—have\nbeen previously reported in various models. The authors should better articulate what is unique here, such as the single-cell\nresolution, the application to clinically relevant HSPCs, and the integration of multiple orthogonal measurements. Clarifying\nhow this method improves on prior genomic instability assessments will strengthen the case for publication.\n2) Utility of scDNA-Seq Platform\nThe custom amplicon panel targets two chromosomes, enabling high-sensitivity detection of on-target LOH but limiting\ninsight into genome-wide effects. The authors should discuss the trade-off between sensitivity and genomic breadth and\nwhether their approach could be adapted to broader panels. The authors should also include a detailed cost estimate.\nIncluding a summary of SNP coverage, read depth, and dropout rates would also help readers assess the platform’s\nrobustness and generalizability.\n3) Role of DDR mechanism\nThe classification of LOH as either copy-neutral or deletion-based is an important strength of the study, but the underlying\nmechanisms (e.g., homologous recombination, break-induced replication, segregation errors) are only briefly discussed.\nAdditional interpretation or speculation on how specific repair pathways contribute to observed LOH patterns—especially in\nthe presence of AZD7648—would add mechanistic depth. Similarly, the authors should explain why palbociclib lowers LOH\nfrequency without altering the LOH subtype distribution.\n4) Applicability\nThe authors suggest their method should be incorporated into clinical genome editing workflows. To support this claim, more\ndiscussion is needed on scalability, throughput, and cost-effectiveness. How many cells must be sequenced to detect a rare\n(e.g., 0.1%) LOH event with confidence? Can this method be used as a release test for edited cell therapies? Addressing\nthese questions would help clarify the translational potential of the approach.\n5) Use of palbociclib\nThe protective effect of palbociclib is encouraging, but the impact on editing efficiency is not reported. If available, data on\nHDR or indel rates in the presence of palbociclib would be valuable to assess the trade-off between genomic safety and\nediting outcomes. Similarly, longer-term consequences of LOH events on cell proliferation and function—particularly in\nHSPCs—should be briefly discussed, even if not directly tested.\nMinor Comments\nSeveral sentences require grammatical correction and improved clarity. The manuscript would benefit from thorough\nproofreading.\nDefine all abbreviations at first use (e.g., PGE, LOH ploidy) and ensure consistency in terminology (e.g., indels vs InDels).\nConsider adding a schematic of the scDNA-seq workflow to aid reader understanding.\nEnsure figure legends clearly explain what is shown (e.g., what “LOH ploidy” values mean) and include appropriate\nstatistical annotations.\nBetter integration of recent literature on CRISPR-induced chromosomal rearrangements and prior\nsingle-cell genotoxicity assays would provide a stronger foundation for the study.", "reviewer_2": "This manuscript describes a technically robust and well-executed study that uses single-cell DNA sequencing (scDNA-seq)\nto examine genotoxicity caused by CRISPR-Cas9 editing in primary human cells, fibroblasts and hematopoietic\nstem/progenitor cells (HSPCs). The authors developed a custom single-nucleotide polymorphism (SNP)-based panel to\ndetect and classify loss of heterozygosity (LOH) and chromosomal rearrangements. They used this panel to uncovered\nmegabase-scale deletions and copy-neutral (CN) LOHs that conventional bulk or single-cell RNA (scRNA)-based methods\nmay miss. The authors then applied this panel to evaluate the detrimental effects of DNA-PK inhibitors, such as AZD7648,\non genome stability and demonstrate the protective effects of the CDK4/6 inhibitor palbociclib in mitigating chromosomal\nrearrangements.\nThe study is technically robust and includes extensive validation. However, as detailed below, the advance is incremental\nand largely confirmatory. The presented findings build upon observations that have already been published, many of which\nare from the same group. Therefore, in this reviewer’s opinion, the work does not meet the novelty threshold required by a\nhigh-profile journal such as Nature Communications.\nA. Key Findings and Prior Art:\n1. CRISPR induces megabase-scale LOH and large rearrangements.\nThis phenomenon is already well established: Kosicki et al., Nat Biotechnol 2018, and Cullot et al., Nat Commun 2019 (from\nthe same group), reported large deletions and complex rearrangements. Boutin et al., Nat Commun 2021 (from the same\ngroup), described CRISPR-Cas9-induced CN-LOH. Leibowitz et al., Nat Genet 2021, and Nahmad et al., Nat Biotechnol\n2022, extended these observations to chromothripsis and aneuploidy.\n2. DNA-PKcs inhibition causes genotoxicity.\nCullot et al., Nat Biotechnol 2024, and Wang et al., Nucleic Acids Res 2024, have already reported that DNA-PKcs inhibitors\npromote megabase-scale deletions and other chromosomal alterations, such as translocations. Thus, the observation here\nis not novel — rather, this is a confirmation of previously published effects using a novel technology (scDNA-seq).\n3. Palbociclib prevents/reduces genotoxicity.\nThis is again an incremental contribution. Cullot et al., Nat Commun 2023 (from the same group), already showed that\npalbociclib reduces CRISPR-induced rearrangements in fibroblasts. The general principle that reducing cell proliferation\nreduces CRISPR genotoxicity has been reported in T cells (Tsuchida et al., Cell 2023) and HSPCs (Zeng et al., Cell Stem\nCell 2025) before.\n4. scDNA-seq as a QC tool.\nThe Tapestri platform has been established for single-cell genotyping (e.g. Ruff et al., Methods Mol Biol 2022). Kalter et al.,\nMol Ther Methods Clin Dev 2025, have built a comprehensive framework for measuring CRISPR editing outcomes,\nincluding off-target activity and chromosomal translocations, using scDNA-seq. Hence, while the authors here emphasize\nsensitivity and resolution of scDNA-seq, the conceptual advance is modest.\nB. Additional Points\n1. While the study has translational intent, the clinical significance of the observed LOH frequencies (e.g., 5–10%) remains\nspeculative. Xenograft experiments to test engraftment and clonal behavior of LOH-containing HSPCs would be important to\nsupport the clinical relevance. Whether LOH leads to dysregulation of imprinted genes, loss of tumor suppressors, or clonal\nexpansion should be tested functionally.\n2. The use of palbociclib to reduce genotoxicity is intriguing. However, the long-term impact on HSPC functon(e.g., self-\nrenewal, multilineage output) is not addressed. Additional phenotypic or engraftment data would strengthen the claim of\nclinical utility.\n3. The study does not include any assessment of off-target genotoxicity, which is an important contributor to CRISPR-\nassociated genomic instability and should at least be discussed.\n4. Several figures rely on low replicate numbers (n = 1–3), and replicate information is sometimes missing. A thorough\nstatistical evaluation of the LOH effects would be desirable."}, "author_response": "POINT by POINT responses to REVIEWERS\nWe would like to thank the reviewers for the time they devoted to this review and for their insightful\ncomments. We have done our best to answer all the questions and comments they made and we\nrecognise that this has improved the quality of the paper. All the modifications are in red in the\nmanuscript. Responses to reviewers in blue after each comment.\nREVIEWER COMMENTS WITH ANSWERS\nReviewer #1:\nMajor Comments\n1) Novelty\nWhile the study is methodologically strong, the novelty of the findings is not clearly delineated.\nSeveral key observations—such as CRISPR-induced LOH, the exacerbating effect of DNA-PKcs\ninhibition, and the protective role of G1 arrest—have been previously reported in various models.\nThe authors should better articulate what is unique here, such as the single-cell resolution, the\napplication to clinically relevant HSPCs, and the integration of multiple orthogonal measurements.\nClarifying how this method improves on prior genomic instability assessments will strengthen the\ncase for publication.\nYou are right, some observations presented in the paper have been previously reported by us and\nothers. However, this study is unique by many points:\n• By the methods. We combined 3 single-cell resolution methods (micronuclei, FAMReD and\nscSNP-DNAseq) to monitor ON-target genotoxicity (and not OFF-target proposed by Kalter et\nal 2025), which is very novel. Importantly, we developed a very innovative scDNAseq\napproach, based on high amplicon frequency close to the DSB and SNP. We compared 2\nbioinformatic methods ( UMAP, fig 2 vs 5 high quality SNP, fig 3). 5 SNP bioinformatic\nmethod allows us to map and compare copy-neutral LOH and copy-loss LOH for each\nexperimental condition with a kinetic of genotoxicity observed. This analysis detects and\nmaps with high sensitivity telomeric megabase-scale and interstitial kilobase-scale\nrearrangements in human clinically-relevant cell for gene therapy. We clarified how our\nmethods improve on prior genomic instability assessments in the text.\n• By the results. For the first time, we observed megabase-scale deletion in HSPCs at day 4\nafter HBG1/2p targeting. We demonstrated palbociclib unknown effect to prevent this\ngenotoxicity in HSPCs.\n• Moreover, to further improve the novelty during the reviewing, we added strong results\nfrom a long study already done in the lab to be separately published. We decided to\nincorporate them in the paper to reinforce it.\nThese data show that:\n• palbociclib is a valuable drug for clinical application: it not only mitigates the\ngenotoxicity but also increases edited HSPCs engraftment.\n• scSNP-DNAseq is a suitable tool to monitor long-term genotoxicity in vivo. For the\nfirst time, we observed that genotoxicity in HSPCs targeting HBG1/2p disappeared at\nday 90 after graft, even if genotoxic AZD7648 is used during editing.\nTo highlight the novelty of our paper, we modified the title, introduction, reorganized the figures and\nthe results and rewrote the discussion (in red in the manuscript).\n2) Utility of scDNA-Seq Platform\nThe custom amplicon panel targets two chromosomes, enabling high-sensitivity detection of on-\ntarget LOH but limiting insight into genome-wide effects. The authors should discuss the trade-off\nbetween sensitivity and genomic breadth and whether their approach could be adapted to broader\npanels. The authors should also include a detailed cost estimate.\nYou're right. It's very important to discuss the clinical benefits of scSNP-DNAseq. We've added to the\ndiscussion elements concerning the method's sensitivity, scalability, genomic coverage, cost, and\npossible future developments.\nIncluding a summary of SNP coverage, read depth, and dropout rates would also help readers\nassess the platform’s robustness and generalizability.\nWe added these global metrics in extended table 4 and in raw data for each sample.\n3) Role of DDR mechanism\nThe classification of LOH as either copy-neutral or deletion-based is an important strength of the\nstudy, but the underlying mechanisms (e.g., homologous recombination, break-induced\nreplication, segregation errors) are only briefly discussed. Additional interpretation or speculation\non how specific repair pathways contribute to observed LOH patterns—especially in the presence\nof AZD7648—would add mechanistic depth. Similarly, the authors should explain why palbociclib\nlowers LOH frequency without altering the LOH subtype distribution.\nTo improve the DDR mechanism understanding, we extended the discussion. Moreover, to decipher\nhow palbociclib lower megabase-scale LOH frequency, we added new experiments. We hypothesized\nthat, by blocking cells in G0/G1 phases, palbociclib could facilitate DSB repair by NHEJ. To test it, we\nco-incubated cells with palbociclib and NHEJ inhibitor during editing. As hypothesized, concomitant\nNHEJ inhibition to palbociclib blocks palbociclib effect demonstrating that its preventing effect was\ndue to higher NEHJ rate that increases chromosome ends binding (supplementary Fig.4)\n4) Applicability\nThe authors suggest their method should be incorporated into clinical genome editing workflows.\nTo support this claim, more discussion is needed on scalability, throughput, and cost-effectiveness.\nHow many cells must be sequenced to detect a rare (e.g., 0.1%) LOH event with confidence? Can\nthis method be used as a release test for edited cell therapies? Addressing these questions would\nhelp clarify the translational potential of the approach.\nYou're right. It's very important to discuss the clinical benefits of scSNP-DNAseq. We've added to the\ndiscussion elements concerning the method's sensitivity, scalability, genomic coverage, cost, and\npossible future developments.\n5) Use of palbociclib\nThe protective effect of palbociclib is encouraging, but the impact on editing efficiency is not\nreported. If available, data on HDR or indel rates in the presence of palbociclib would be valuable\nto assess the trade-off between genomic safety and editing outcomes.\nPalbociclib does not reduce CRISPR-efficiency and InDels/deletion frequency is not modulated or\nreduced by palbociclib. We added the InDels rate and genomic profile after editing for each\nexperiment with palbociclib (for example supplementary Fig 1, using Nanopore sequencing).\nJust to clarify, Palbociclib blocks the cells in G0/G1 phase to reduce genotoxicity. Because HDR/PGE is\nS/G2/M phase-dependent, the drug can only be used to invalidate genes by NHEJ.\nWe also reinforced results on the palbociclib effect on HSPCs by characterizing its effect on\nproliferation, cell death, stemness profile and graft capability (Fig 4 and supplementary Fig 5).\n6) longer-term consequences of LOH events on cell proliferation and function—particularly in\nHSPCs—should be briefly discussed, even if not directly tested.\nYou are right, longer-term consequences of LOH events, in particular in HSPCs, are critical for the\ngene therapy field. We tested it. To test longer-term consequences of LOH events on HSPCs\nproliferation and function, we edited HSPCs by CRISPR at HBG1/2 p (chr11p). We grafted edited\nHSPCs in 2 mice per condition (without or with AZD7648). scSNP-DNA seq revealed that cells with\nLOH disappear after 90 days graft in vivo, suggesting that, fortunately, LOH events don’t give a\nselective advantage ( even with pre-incubation with genotoxic AZD7648). No genotoxicity was\ndetected, even with a high sensitive single cell resolution method. .\nThese results are in accordance with the disappearance of the rearranged CRISPR-CAR-T cells after in\nvitro 14 days-culture (Nahmad et al) and after in vivo in patients (Tsuchida et al). We added these\nimportant novel results in the manuscript and discussed them (new Figure 7).\nMinor Comments\nSeveral sentences require grammatical correction and improved clarity. The manuscript would\nbenefit from thorough proofreading.\nWe tried to improve english sentences and clarity.\nDefine all abbreviations at first use (e.g., PGE, LOH ploidy) and ensure consistency in terminology\n(e.g., inDels vs InDels).\nDone\nConsider adding a schematic of the scDNA-seq workflow to aid reader understanding.\nWe modified experimental designs at the beginning of each figure to be clearer.\nEnsure figure legends clearly explain what is shown (e.g., what “LOH ploidy” values mean) and\ninclude appropriate statistical annotations.\nWe improved figure legends (“Ploidy in LOH genomic area was calculated by the mean ratio of read\ndepth of amplicons in the LOH area of edited LOH cells to non-edited cells”) and included statistical\ntests for each figure.\nBetter integration of recent literature on CRISPR-induced chromosomal rearrangements and prior\nsingle-cell genotoxicity assays would provide a stronger foundation for the study.\nWe added 11 recent publications for a stronger foundation for the study (in red). Thanks for this\nadvice.\nReviewer #2 (Remarks to the Author):\nThe study is technically robust and includes extensive validation. However, as detailed below, the\nadvance is incremental and largely confirmatory. The presented findings build upon observations\nthat have already been published, many of which are from the same group. Therefore, in this\nreviewer’s opinion, the work does not meet the novelty threshold required by a high-profile\njournal such as Nature Communications.\nA. Key Findings and Prior Art:\n1. CRISPR induces megabase-scale LOH and large rearrangements.\nThis phenomenon is already well established: Kosicki et al., Nat Biotechnol 2018, and Cullot et al.,\nNat Commun 2019 (from the same group), reported large deletions and complex rearrangements.\nBoutin et al., Nat Commun 2021 (from the same group), described CRISPR-Cas9-induced CN-LOH.\nLeibowitz et al., Nat Genet 2021, and Nahmad et al., Nat Biotechnol 2022, extended these\nobservations to chromothripsis and aneuploidy.\n2. DNA-PKcs inhibition causes genotoxicity.\nCullot et al., Nat Biotechnol 2024, and Wang et al., Nucleic Acids Res 2024, have already reported\nthat DNA-PKcs inhibitors promote megabase-scale deletions and other chromosomal alterations,\nsuch as translocations. Thus, the observation here is not novel — rather, this is a confirmation of\npreviously published effects using a novel technology (scDNA-seq).\n3. Palbociclib prevents/reduces genotoxicity.\nThis is again an incremental contribution. Cullot et al., Nat Commun 2023 (from the same group),\nalready showed that palbociclib reduces CRISPR-induced rearrangements in fibroblasts. The\ngeneral principle that reducing cell proliferation reduces CRISPR genotoxicity has been reported in\nT cells (Tsuchida et al., Cell 2023) and HSPCs (Zeng et al., Cell Stem Cell 2025) before.\n4. scDNA-seq as a QC tool.\nThe Tapestri platform has been established for single-cell genotyping (e.g. Ruff et al., Methods\nMol Biol 2022). Kalter et al., Mol Ther Methods Clin Dev 2025, have built a comprehensive\nframework for measuring CRISPR editing outcomes, including off-target activity and chromosomal\ntranslocations, using scDNA-seq. Hence, while the authors here emphasize sensitivity and\nresolution of scDNA-seq, the conceptual advance is modest.\nYou are right, some observations presented in the paper have been previously reported by us and\nothers. However, this study is unique by many points:\n• By the methods. We combined 3 single-cell resolution methods (micronuclei, FAMReD and\nscSNP-DNAseq) to monitor ON-target genotoxicity, in particular LOH (and not OFF-target and\ntranslocations proposed by Kalter et al 2025), which is very novel. Importantly, we developed\na very innovative scDNAseq approach, based on high amplicon frequency close to the DSB\nand SNP to quantify non detected copy neutral LOH. We compared 2 bioinformatic methods\n(UMAP, fig 2 vs 5 high quality SNP, fig 3). 5 SNP bioinformatic method allows us to map and\ncompare copy-neutral LOH and copy-loss LOH for each experimental condition with a kinetic\nof genotoxicity observed. This analysis detects and maps with high sensitivity telomeric\nmegabase-scale and interstitial kilobase-scale rearrangements in human clinically-relevant\ncells for gene therapy. We clarified how our methods improve on prior genomic instability\nassessments in the text.\n• By the results. For the first time, we observed megabase-scale deletion in HSPCs at day 4\nafter HBG1/2p targeting. We demonstrated palbociclib unknown effect to prevent this\ngenotoxicity in HSPCs.\n• Moreover, to further improve the novelty during the reviewing, we added strong results\nfrom a long study already done in the lab to be separately published. We decided to\nincorporate them in the paper to reinforce it.\nThese data show that:\n• palbociclib is a valuable drug for clinical application: it not only mitigates the\ngenotoxicity but also increases edited HSPCs engraftment.\n• scSNP-DNAseq is a suitable tool to monitor long-term genotoxicity in vivo. For the\nfirst time, we observed that genotoxicity in HSPCs targeting HBG1/2p disappeared at\nday 90 after graft, even if genotoxic AZD7648 is used during editing.\nTo highlight the novelty of our paper, we modified the title, introduction, reorganized the figures and\nthe results and rewrote the discussion (in red in the manuscript). We hope that you can consider that\nnovelty is enough with these important improvements.Our study strongly complements the large\nliterature on genotoxicity due to CRISPR-Cas9-induced DSB. All the references cited by teh reviewer\n#2 are in the manuscript.\nB. Additional Points\n1. While the study has translational intent, the clinical significance of the observed LOH\nfrequencies (e.g., 5–10%) remains speculative. Xenograft experiments to test engraftment and\nclonal behavior of LOH-containing HSPCs would be important to support the clinical relevance.\nWhether LOH leads to dysregulation of imprinted genes, loss of tumor suppressors, or clonal\nexpansion should be tested functionally.\nYou are right, longer-term consequences of LOH events, in particular in HSPCs, are critical for the\ngene therapy field. We tested it. To test longer-term consequences of LOH events on HSPCs\nproliferation and function, we edited HSPCs by CRISPR at HBG1/2 p (chr11p). We grafted edited\nHSPCs in 2 mice per condition (without or with AZD7648). scSNP-DNA seq revealed that cells with\nLOH disappear after 90 days graft in vivo, suggesting that, fortunately, LOH events don’t give a\nselective advantage ( even with pre-incubation with genotoxic AZD7648). No genotoxicity was\ndetected, even with a high sensitive single cell resolution method.\nThese results are in accordance with the disappearance of the rearranged CRISPR-CAR-T cells after in\nvitro 14 days-culture (Nahmad et al) and after in vivo in patients (Tsuchida et al). We added these\nimportant novel results in the manuscript and discussed them (new Figure 7).\n2. The use of palbociclib to reduce genotoxicity is intriguing.\nWe hypothesized that, by blocking cells in G0/G1 phases, palbociclib could facilitate DSB repair by\nNHEJ. To test it, we co-incubated cells with palbociclib and NHEJ inhibitor during editing. As\nhypothesized, concomitant NHEJ inhibition to palbociclib blocks palbociclib effect demonstrating that\nits preventing effect was due to higher NEHJ rate that increases chromosome ends binding\n(supplementary Fig.4).\nHowever, the long-term impact on HSPC function(e.g., self-renewal, multilineage output) is not\naddressed. Additional phenotypic or engraftment data would strengthen the claim of clinical\nutility.\nFor palbociclib clinical translation, it is important to test its impact on HSPCs behaviour. Interestingly,\nwe added results on the palbociclib effect on HSPCs by characterizing its effect on proliferation,\ncell death, stemness profile and graft capability (Fig 4 and supplementary Fig 5). Our data demonstrate\nfor the first time that palbociclib is a valuable drug for clinical application: it not only mitigates the\ngenotoxicity but also increases edited HSPCs engraftment.\n3. The study does not include any assessment of off-target genotoxicity, which is an important\ncontributor to CRISPR-associated genomic instability and should at least be discussed.\nOFF-target genotoxicity is the best known and documented. A recent study showed the value of\nscDNAseq for its sensitive analysis at predicted OFF-target sites (Kalter et al 2025). To avoid any\nanalysis bias, we used a high-fidelity Cas9 and chose targets for which no or few OFF-targets are\ndescribed ( cf Sharma et al for HBG1/2p targeting and Cullot et al 2019 for UROS). Our originality lies\nin our focus on ON-target genotoxicity and, in particular, major kilo and megabasic rearrangements.\nOur focus on SNPs enables us for the first time to study the often underestimated copy-neutral\nLOH. In any case, it is highly relevant to study ON and OFF-target genotoxicity at the same time.\nInterestingly, scSNPDNAseq is an appropriate method. New primer design would enable this analysis.\nWe have added these elements to the discussion.\n4. Several figures rely on low replicate numbers (n = 1–3), and replicate information is sometimes\nmissing. A thorough statistical evaluation of the LOH effects would be desirable.\nWe completed the data, indicated the replicate numbers, and the statistical test used for all\nexperiments."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed my main concerns and questions and the paper is suitable for publication.", "reviewer_2": "The authors have substantially strengthened the manuscript in revision. They now provide important new data, including in\nvivo evidence that LOH events in HSPCs disappear after transplantation, mechanistic experiments demonstrating that\npalbociclib’s protective effect is NHEJ-dependent, and functional studies showing improved engraftment of edited HSPCs.\nTogether, these additions significantly enhance the clinical relevance and translational potential of the study.\nWhile several of the central observations build on prior work from this and other groups, the combination of three orthogonal\nsingle-cell assays and the development of a high-sensitivity scSNP-DNAseq platform represent meaningful methodological\nprogress. The integration of long-term safety data in clinically relevant cells further increases the impact.\nHowever, several concerns remain:\n• Clarity and language: Although the figures are now more accessible and the data more clearly described, the manuscript\nstill requires substantial improvement in English language and style. In particular, the descriptions of figures are often\ndifficult to follow. The text must be revised to provide a clearer explanation of how experiments were conducted, how data\nwere obtained, and what is shown in the figures. Too much is left implicit, forcing the reader to guess.\n• Figure organization: The current sequence is suboptimal. I would recommend starting with Figures 5 and 6, followed by\nFigures 1–4 and 7. The in vivo data are currently split between Fig. 4f–h and Fig. 7, which makes little sense. These data\nshould be consolidated into 1–2 figures. Most importantly, the effect of palbociclib on LOH should also be demonstrated in\nvivo, which is absolutely crucial.\n• Scope of findings: The conclusions are largely based on HBG targeting. This limitation should be explicitly acknowledged,\nas results may be target-specific and not necessarily generalizable.\nSpecific points:\n• Specify consistently throughout the manuscript, including the title, that genotoxicity is defined as on-target genotoxicity,\nsince off-target effects were not assessed.\n• Abstract: The sentence “Fortunately, targeting Chr11p, scSNP-DNA-seq revealed that genotoxic events disappeared after\nlong-term xenografts, even using AZD7648” could be misleading. While the genotoxic events disappear in this particular\nsetting/experiment and also in other examples referenced by the authors, it should not be generalized. It could well be that\nsuch genotoxic events do not disappear if another locus is targeted. This sentence should be amended or deleted from the\nabstract.\n• Introduction, page 3, 2nd section: Why introduce a new abbreviation, PGE? HDR is a well-accepted term in the field.\n• Introduction, page 4, last section: Consider mentioning the in vivo experiments already in this summary in the introduction.\n• Figure 2: Clarify the meaning of colors in Fig. 2b (blue/pink/white): It says blue stands for WT, white for HETerozygous and\npink for HOMozygous. In the text the authors state however “In these cells, 18 SNPs located between the HBG1/2p cut sites\nand the Chr11p telomere were lost (only pink or only blue, Fig. 2b right)”, suggesting that pink and blue stand for allelic variantd.\nWhat is correct?\n• Figure 3: What is the relationship between the 5–7 SNPs in Fig. 3 and the 18 SNPs in Fig. 2? What does “more reliable\namplicons” in the main text with reference to these new amplicons mean?\n• Figure 3: The legend redundancies and inconsistencies in the figure must be corrected. Why do the authors present two\nlegends for these 7 new SNP amplicons (panels c and f, one is sufficient in my view)? Fig. 3d does not show SNP6 and\nSNP7, as stated in the text. Also, clarify whether the same DNA samples were analyzed across figures.\n• Figures 3g: Clarify which amplicons were used. I can see 13-14 amplicons to the left of the target site, so it’s neither 7 as in\nFig. 3f nor 18 as in Fig. 2c. What do the different dot colors mean?\n• Figures 3g: Clarify what percentage the profiles “Termin LOH” and “interstitial LOH” represent: is it 129 or 38 out of 1855,\nrespectively?\n• Figures 3g: Typographical errors should be corrected.\n• Supplementary Fig. 2: Include the missing palbociclib data to support the comparison with cytokine starvation.\n• Figure 4c: The authors state: “HBG1/2p targeting by CRISPR induced a high rate of abnormal nucleus, hallmarks of the\nelimination of amplified DNA and/or DNA repair complexes, reduced by palbociclib use during editing (Fig. 4c left).” Provide\nmethodological details (e.g., microscopy, number of cells analyzed).\n• Figure 4d: Clarify what amplicon panel was used. Also consider testing low cytokine concentrations (e.g. 3 ng/mL) as a\ncomparison to palbociclib.\n• Supplementary Fig. 4: The phrasing “genotoxicity was rescued” is misleading; the data show aggravation. Moreover, data\ncombining cell cycle inhibition with NHEJ inhibition are missing in Fig. S4c.\n• Figure 4f: Rephrase in the main text to “…palbociclib exposure led to a relative increase of the most primitive stem cell\npopulation” as it is not clear whether this reflects true expansion.\n• Figure 4f-h: These in vivo experiments are very important and add to the value of the manuscript. However, it is absolutely\ncrucial to include the HBG-edited HSPCs into these datasets. Some of these results are currently shown in Fig. S5 and\nshould be transferred to Fig. 4 (while Fig. 4h could be transferred to suppl. data). However, it is absolutely crucial to show\nthe long-term effects of palbociclib on LOH also in vivo.\n• Figure 5: The authors conclude “Compared to other HDR boosters, AZD7648 reached the highest HDR rate but also the\nhighest shortterm genotoxicity in hFFs.” As recently published and as known to the authors, measuring HDR frequencies by\nshort-read amplicon NGS can be misleading. This should be clarified in the text and especially in the conclusions.\n• Figures 5–6: These experiments currently feel disconnected from the main narrative. Consider reordering. E.g., start with\nNHEJ inhibition as a negative example, then introduce palbociclib as a mitigation strategy (see general remark above)."}, "author_response": "Thank you for your positive comments. We agree that the new data significantly enhance the\nlevel of the publication and the clinical relevance. In vivo data, palbociclib mechanism and\nimprovement of edited HSPCs engraftment are now central to the work. We modified the\nfigures organisation.\nTo give pride of place to new results, we merged both AZD7648 figures, transferred Fig S5 to\nin vivo data of Fig 4, and proposed a new organization of the manuscript. We hope that\nchanges are in accordance with your comments.\nSee point by point response to your remaining concerns:\n1\n• Clarity and language: Although the figures are now more accessible and the data more\nclearly described, the manuscript still requires substantial improvement in English language\nand style. In particular, the descriptions of figures are often difficult to follow.\nThe text must be revised to provide a clearer explanation of how experiments were\nconducted, how data were obtained, and what is shown in the figures. Too much is left\nimplicit, forcing the reader to guess.\nWe improved our English language and style and we edited the figures description (legends\nand results) to facilitate manuscript reading. We hope it is now clearer.\n• Figure organization: The current sequence is suboptimal. I would recommend starting with\nFigures 5 and 6 = AZD, followed by Figures 1–4 and 7. The in vivo data are currently split\nbetween Fig. 4f–h and Fig. 7, which makes little sense. These data should be consolidated\ninto 1–2 figures. Unfortunately, we can not start with figures 5 and 6 because\nwe first need to validate our scSNPDNA seq et bioinformatic pipelines (Fig 1/2/3). As\nsuggested, because palbociclib and in vivo data are more important than AZD7648, Figures\n5+6 with AZD7648 were merged in a new Fig. 4, and new Fig 5/6/7 are dedicated to palbociclib\nand in vivo consolidated data.\nMost importantly, the effect of palbociclib on LOH should also be demonstrated in vivo,\nwhich is absolutely crucial.\nYou are right, we did not perform scSNP-DNA seq in vivo to assess long-term palbociclib effect\non LOH. Indeed, even targeting HBG1/2p, the worst genotoxic CRISPR target (due to double\ncuts) and the use of deleterious AZD7648, we did not detect LOH in mice 90-days post graft.\nConsidering the stronger protective effect of palbociclib, (15-fold decrease of LOH compared\nto AZD7648), there is no reason to think that we will find any LOH in mice after palbociclib\ntreatment. But indeed, this long and expensive experiment could be very instructive if CRISPR\ninduces persistent LOH in mice. Further studies will be required to test another loci. We added\nthis point in the discussion.\n2\n• Scope of findings: The conclusions are largely based on HBG targeting. This limitation should\nbe explicitly acknowledged, as results may be target-specific and not necessarily generalizable.\nYou are right. We added this point in discussion. “After HBG1/2p targeting, ON-target\ngenotoxicity disappears in vivo 3 months after engraftment. This result cannot be generalized,\nbecause cell consequences of chromosomal rearrangement are probably targeted locus-\ndependent.”\nSpecific points:\n• Specify consistently throughout the manuscript, including the title, that genotoxicity is\ndefined as on-target genotoxicity, since off-target effects were not assessed.\nWe clarified this point, in the title and in the manuscript.\nThe new title is “Single-cell multiplex approaches deeply map ON-target CRISPR-genotoxicity\nand reveal its mitigation by palbociclib and long-term engraftment”\n• Abstract: The sentence “Fortunately, targeting Chr11p, scSNP-DNA-seq revealed that\ngenotoxic events disappeared after long-term xenografts, even using AZD7648” could be\nmisleading. While the genotoxic events disappear in this particular setting/experiment and\nalso in other examples referenced by the authors, it should not be generalized. It could well\nbe that such genotoxic events do not disappear if another locus is targeted. This sentence\nshould be amended or deleted from the abstract.\nYou are right, it is probably locus-dependent. We modified the abstract to be more precise.\n“Fortunately, targeting HBG1/2p in Chr11p in HSPCs, scSNP-DNA-seq revealed that ON-target\ngenotoxic events disappeared after long-term xenografts, even using AZD7648”. We also\nadded this point in discussion, in the paragraph “limitations”.\n• Introduction, page 3, 2nd section: Why introduce a new abbreviation, PGE? HDR is a well-\naccepted term in the field.\nPrecise genome editing is also used in CRISPR-field. To simplify the manuscript, we now only\nuse HDR.\n3\n• Introduction, page 4, last section: Consider mentioning the in vivo experiments already in\nthis summary in the introduction.\nIt is already mentioned “we monitored it in vivo”. We modified by “ to monitor it persistence\nin vivo after engraftment” to be clearer.\n• Figure 2: Clarify the meaning of colors in Fig. 2b (blue/pink/white): It says blue stands for WT, white for HETerozygous and\npink for HOMozygous. In the text the authors state however “In these cells, 18 SNPs located between the HBG1/2p cut sites\nand the Chr11p telomere were lost (only pink or only blue, Fig. 2b right)”, suggesting that pink and blue stand for allelic variantd.\nWhat is correct?\nWe tried to clarify. For that, we modified the legend and the description of Fig 2b.\nBriefly, In chr11p, centromeric to the cut, most of SNPs are white = heterozygous non edited\nSNP = A/B. In LOH cells, telomeric to the cut, all the 18 SNPs became homozygous: A/A (pink)\nor B/B (blue).\nWe hope it is now clearer.\n• Figure 3: What is the relationship between the 5–7 SNPs in Fig. 3 and the 18 SNPs in Fig.\n2? What does “more reliable amplicons” in the main text with reference to these new\namplicons mean?\nIn Fig. 2, all SNPs present in the unedited cells are used to cluster the cells on their SNP profiles\n(global UMAP analysis based on 88 SNPs). In Fig 3, only amplicons containing 5 high-quality\nselected SNPs (a panel of 5 discriminant SNPs) are used. Obviously, they are part of those used\nin Fig 2. Quality criteria for these selected SNP is detailed in the text. This information was\nadded in the text.\n• Figure 3: The legend redundancies and inconsistencies in the figure must be corrected.\nWhy do the authors present two legends for these 7 new SNP amplicons (panels c and f, one\nis sufficient in my view)? Fig. 3d does not show SNP6 and SNP7, as stated in the text. Also,\nclarify whether the same DNA samples were analyzed across figures.\nThank you for your attentive review. We designed two panels with 5 SNPs. The first, designed\nto detect megabasic events with SNP 1-5 and the second, to detect kilobasic and megabasic\n4\nevents with SNP 1/2/6/7/5. The results obtained with both panels were from the same edited\ncells. We clarify that in the text and in the figure.\n• Figures 3g: Clarify which amplicons were used. I can see 13-14 amplicons to the left of the\ntarget site, so it’s neither 7 as in Fig. 3f nor 18 as in Fig. 2c. What do the different dot colors\nmean? Clarify what percentage the profiles “Termin LOH” and “interstitial LOH” represent:\nis it 129 or 38 out of 1855, respectively?\nBriefly, we first clustered the cell populations based on the presence or absence of LOH: WT,\nterminal LOH profile 1, terminal LOH profile 2, etc., using either all SNPs present in the\nunedited cells (UMAP, 88 SNPs, Fig. 2) or only 5 high-quality SNPs (Figs. 3–7). After clustering,\nCNV analysis was performed by considering all high-quality amplicons. Ploidy was estimated\nusing the read-depth of amplicons located in the LOH regions of cells, normalized to the read-\ndepth of the same amplicons in normal cells. For each cell in each population, LOH ploidy was\ncalculated as the mean ploidy of amplicons within the LOH genomic region. Using this\napproach, 13–14 amplicons located to the left of the target site were selected based on quality\ncriteria (as defined in the Methods section).\nWe revised the manuscript to include additional methodological details in both the Results\nand Methods sections to improve clarity.\nRegarding the dot colors (Fig. 3g, right panel), in the manuscript, telomeric LOH I and LOH II\nare consistently represented in dark pink and dark blue, respectively, while interstitial LOH I\nand LOH II are shown in pink and blue. A color gradient is used for each LOH type to distinguish\nbetween copy-neutral and copy-loss LOH.\nConcerning Fig. 3g, you are right, 1855 cells were correctly sequenced. Among them, 129 cells\nexhibited a terminal 11p LOH CNV ( 67 + 62), histogram detailed the proportion of copy-\nneutral and copy-loss profiles (80% had a copy-loss LOH and 20% a copy-neutral LOH). In the\nsame way, 38 among the 1855 analyzed cells exhibited an interstitial LOH (19 + 19) and\nhistogram detailed the proportion of copy-neutral and copy-loss profiles.\nWe added the number of cells in the histograms and modified the results to be clearer.\n• Figures 3g: Typographical errors should be corrected.\n• Supplementary Fig. 2: Include the missing palbociclib data to support the comparison with\ncytokine starvation.\nWe did it to facilitate comparison. See new Fig. S9.\n• Figure 4c: The authors state: “HBG1/2p targeting by CRISPR induced a high rate of\nabnormal nucleus, hallmarks of the elimination of amplified DNA and/or DNA repair\ncomplexes, reduced by palbociclib use during editing (Fig. 4c left).” Provide\nmethodological details (e.g., microscopy, number of cells analyzed).\nNumber or analyzed cells for DAPI staining is now mentioned in the legend. We added the\nnumber of analyzed cells for cytometry in Figure S5 b and c and Fig 4 and 5.\nWe also precise “fluorescence” microscopy in the legend and in the text.\n• Figure 4d: Clarify what amplicon panel was used.\nFigure 4d is now Figure 5d. For scSNP-DNAseq analysis, kilobasic Panel SNP (1/2/6/7/5) was\nused (Fig. 3f) to detect interstitial and telomeric LOHs. We added this important information\nin the text, thank you.\n● Also consider testing low cytokine concentrations (e.g. 3 ng/mL) as a comparison to\npalbociclib.\nOur goal was to arrest HSPCs in G0/G1 phases without toxicity.\nComparison between palbociclib vs cytokine starvation revealed that :\n- Palbociclib is more efficient than 3 ng/mL cytokine starvation to synchronize the cells\n(Fig. S9b 36h).\n- Complete cytokine starvation (0 ng/mL) is equally potent to palbociclib to arrest the\ncells in G0/G1 (Fig. S9b 36h) and block proliferation (Fig. S9c) but more toxic (Fig. S9d).\nThat’s why we decided to favor palbociclib, the best protocol, for the following\nexperiments, in particular expensive scDNA seq.\nWe added these points in the text.\n6\n• Supplementary Fig. 4: The phrasing “genotoxicity was rescued” is misleading;\nthe data show aggravation.\nThank you for the comment, we modified the text\nMoreover, data combining cell cycle inhibition with NHEJ inhibition are missing in Fig. S4c.\nIt is an interesting idea. We did the experiment and analyzed the ratio MMEJ/NHEJ profile for\ncombined cell cycle and NHEJ inhibition at day 6 (Nanopore sequencing, Mean+/- SEM, N=3).\nHowever, the drug combination induced early cell death and long-term genotoxicity, as\ndemonstrated by FAMRED at day 18. Early cell death can be explained by the fact that DSB\ncannot be repaired. Indeed, in the G0/G1 phase cycle, the only repair pathway available is\nNHEJ (MMEJ only in S G2M, ref Yu W et al Nature com 2020). In the presence of palbociclib\nand AZD7648, no repair pathways are available, inducing cell death. Moreover, short read\namplicons to analyse frequencies of Indels can be misleading in case of high megabasic\ngenotoxicity and are not appropriate. Taken together, we cannot include this biased data in\nthe manuscript.\n• Figure 4f: Rephrase in the main text to “…palbociclib exposure led to a relative increase of\nthe most primitive stem cell population” as it is not clear whether this reflects true\nexpansion.\nAfter 36h of palbociclib, the number of cells is not increased (fig 6b). Only the proportion of\nstem cell population is more prevalent, suggesting that it is not a true expansion but rather a\nstem cell maintenance/selection or progenitor dedifferentiation. We carried out RTqPCR and\nobserved induced expression of hematopoietic stemness genes (CXCR4, TAL1 and RUNX1, new\n7\nresults added in Fig. 6f) in palbociclib-exposed HSPCs. These new data confirm that palbociclib\nhad a protective/benefic effect on stem cell compartment.\n• Figure 4f-h: These in vivo experiments are very important and add to the value of the\nmanuscript. However, it is absolutely crucial to include the HBG-edited HSPCs into these\ndatasets. Some of these results are currently shown in Fig. S5 and should be transferred to\nFig. 4 (while Fig. 4h could be transferred to suppl. data). However, it is absolutely crucial to\nshow the long-term effects of palbociclib on LOH also in vivo.\nWe followed your advice and modified the figure organization to highlight HBG1/2p-edited\nHSPCs results in vivo, after palbociclib exposure.\nWe agree that long-term protective effect of palbociclib on LOH in vivo is important. To\nvalidate palbociclib effect in vivo, it will be required to find a CRISPR target that induces\nsignificant and persistent in vivo ON-target genotoxicity after graft. We hypothesize that ON-\ntarget genotoxicity is locus dependent, and we are screening genomic areas inducing long-\nterm in vivo genotoxicity, very useful to further evaluate protective effect of palbociclib on\nLOH. In this study, targeting HBG1/2p, even with AZD 7648, highly genotoxic, no LOH were\ndetected 17-week after graft. Therefore, it is unlikely that we observe LOH using palbociclib,\nconsidering its preventing effect already observed in vitro. \n• Figure 5: The authors conclude “Compared to other HDR boosters, AZD7648 reached the\nhighest HDR rate but also the highest short term genotoxicity in hFFs.” As recently published\nand as known to the authors, measuring HDR frequencies by short-read amplicon NGS can\nbe misleading. This should be clarified in the text and especially in the conclusions.\nYou are right, there is a bias in HDR rate analysis. We modified the sentence by\n“Compared to other HDR boosters, AZD7648 reached the highest short-term genotoxicity in\nhFFs.”\nWe added “Importantly, these genotoxicity can produce a bias of editing analysis by short-\nread amplicon, raising the question of the true efficacy of these HDR boosters.” in discussion\nand cited the review C Aussel Nat com 2025\n8\n• Figures 5–6: These experiments currently feel disconnected from the main narrative.\nConsider reordering. E.g., start with NHEJ inhibition as a negative example, then introduce\npalbociclib as a mitigation strategy (see general remark above).\nThank you for this remark. We modified the figure order. We expect that the narrative is now\nappropriate.\n9"}]

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