High-Throughput Imaging of CRISPR- and Recombinant Adeno-Associated Virus-Induced DNA Damage Response in Human Hematopoietic Stem and Progenitor Cells.
CRISPR J
; 5(1): 80-94, 2022 02.
Article
em En
| MEDLINE
| ID: mdl-35049367
ABSTRACT
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Dependovirus
/
Edição de Genes
Tipo de estudo:
Guideline
/
Risk_factors_studies
Limite:
Humans
Idioma:
En
Revista:
CRISPR J
Ano de publicação:
2022
Tipo de documento:
Article
País de afiliação:
Israel