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

Abstract

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