High-throughput Imaging of CRISPR- and Recombinant Adeno-associated Virus-induced DNA Damage Response in Human Hematopoietic Stem and Progenitor Cells

CRISPR-Cas technology has revolutionized gene editing, but concerns remain due to its propensity for off-target interactions. This, combined with genotoxicity related to both CRISPR-Cas9-induced double-strand breaks and transgene delivery, poses a significant liability for clinical genome-editing applications. Current best practice is to optimize genome-editing parameters in preclinical studies. However, quantitative tools that measure off-target interactions and genotoxicity are costly and time-consuming, limiting the practicality of screening large numbers of potential genome-editing reagents and conditions. Here, we show that flow-based imaging facilitates DNA damage characterization of hundreds of human hematopoietic stem and progenitor cells per minute after treatment with CRISPR-Cas9 and recombinant adeno-associated virus serotype 6. With our web-based platform that leverages deep learning for image analysis, we find that greater DNA damage response is observed for guide RNAs with higher genome-editing activity, differentiating even single on-target guide RNAs with different levels of off-target interactions. This work simplifies the characterization and screening process of genome-editing parameters toward enabling safer and more effective gene-therapy applications.

Automated summary

CRISPR-Cas technology has brought revolutionary changes to gene editing, but concerns about off-target interactions and genotoxicity remain. The current practice of optimizing genome-editing parameters in preclinical studies is expensive and time-consuming. However, this study introduces a flow-based imaging method coupled with deep learning for image analysis to characterize DNA damage. The findings show that guide RNAs with higher genome-editing activity induce greater DNA damage response, even differentiating single on-target guide RNAs with varying levels of off-target interactions. This simplifies the process of evaluating and screening genome-editing parameters, enabling safer and more effective gene therapy applications.