PRJNA762559
High-throughput imaging of CRISPR- and rAAV-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 double-strand breaks more generally as well as transgene delivery, pose significant liabilities 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 the DNA damage response correlates with 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.