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.
Bibliographical noteFunding Information:
This work was supported by the European Research Council (ERC) under the European Union Horizon 2020 research and innovation program (grant no. 802567, Y.S.; grant no. 755758, A.H.), the Israel Innovation Authority through the CRISPR-IL consortium, the Zuckerman foundation, and the POLAK Fund for Applied Research at the Technion (Y.S. and L.E.W.).
© Daniel Allen, et al. 2022; Published by Mary Ann Liebert, Inc. 2022.