The use of CRISPR-Cas9 genome editing has been wildly successful in the cell & gene therapy space, but DNA double-strand breaks can trigger severe genotoxicity, including unpredictable chromosomal rearrangements and megabase-scale instability. In this live session, Julian Boutin details how his team deployed a highly sensitive single-cell sequencing approach focused on single nucleotide polymorphisms (scSNP-DNAseq) to deeply map these unintended genomic outcomes after CRISPR gene editing targeting therapeutically relevant loci, such as the HBG1/2 promoters in primary hematopoietic stem cells. By simultaneously tracking hundreds of SNPs and copy number variations at the single-cell level, the researchers successfully quantified and characterized complex loss of heterozygosity (LOH) events.

Discover how this scSNP-DNAseq strategy accurately maps CRISPR-mediated genotoxicity events like chromosomal instability and LOH events, evaluates the exacerbating effects of DNA repair modulators, and validates mitigation strategies like transient cell cycle arrest with palbociclib to ensure the long-term safety of CRISPR-edited therapies.

Key Learnings

• See how scSNP-DNAseq accurately detects and maps rare, CRISPR-induced chromosomal rearrangements and structural genotoxicity in primary human cells.
• Learn to leverage single-cell SNP and copy number analysis to map megabase-scale chromosomal instability and loss of heterozygosity.
• Understand how longitudinal single-cell profiling validates safety interventions, such as palbociclib treatment, and monitors the in vivo persistence of genotoxic events.