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

Area of Science:

• Genetics and Genomics
• Molecular Biology
• Gene Therapy

Background:

• CRISPR-Cas9 nuclease technology offers significant potential for gene therapy applications.
• Ensuring the safety of CRISPR-Cas9 editing, particularly concerning on-target genotoxicity and large-scale genomic rearrangements, is critical.
• Current methods for monitoring CRISPR-induced genotoxicity require improvement in sensitivity and scope.

Purpose of the Study:

• To develop and validate a sensitive, multiplexed approach for monitoring CRISPR-Cas9-mediated genotoxicity in primary cells.
• To detect, map, and characterize on-target genomic rearrangements, including losses of heterozygosity and chromosomal instability.
• To evaluate the efficacy of therapeutic interventions in mitigating CRISPR-associated genotoxicity.

Main Methods:

• A combined single-cell resolution approach integrating single nucleotide polymorphism (SNP) DNA sequencing (scSNP-DNAseq), micronuclei assays, and loss of heterozygosity (LOH) cytometry-reporter assays.
• Application of the developed strategy to primary cells to assess CRISPR-induced genotoxicity.
• Utilizing scSNP-DNAseq to monitor on-target genotoxic events in vivo following long-term xenografts.

Main Results:

• The developed multiplexed strategy enables sensitive detection of CRISPR-mediated genotoxicity in primary cells.
• Various types of induced losses of heterozygosity and editing-associated chromosomal instability were detected, mapped, and characterized.
• Palbociclib demonstrated a protective effect against genomic rearrangements in hematopoietic stem cells without compromising cell function or graft capability.
• The DNA-PKcs inhibitor AZD7648 was found to significantly increase short-term risk.
• On-target genotoxic events were undetectable after long-term xenografts when targeting HBG1/2p, as revealed by scSNP-DNA-seq.

Conclusions:

• The developed scSNP-DNAseq combined approach is a sensitive and effective tool for monitoring CRISPR-mediated genotoxicity.
• Palbociclib shows promise in preventing CRISPR-induced genomic rearrangements, contributing to safer gene therapies.
• Routine implementation of scSNP-DNAseq is recommended for monitoring chromosomal rearrangements before and after CRISPR-edited cell infusions to ensure therapeutic safety.