Exonuclease editor promotes precision of gene editing in mammalian cells.
DMD
Exonuclease editor
Gene correction
Homology-directed repair
Microhomology-based precise deletion
Journal
BMC biology
ISSN: 1741-7007
Titre abrégé: BMC Biol
Pays: England
ID NLM: 101190720
Informations de publication
Date de publication:
20 May 2024
20 May 2024
Historique:
received:
28
04
2023
accepted:
13
05
2024
medline:
21
5
2024
pubmed:
21
5
2024
entrez:
20
5
2024
Statut:
epublish
Résumé
Many efforts have been made to improve the precision of Cas9-mediated gene editing through increasing knock-in efficiency and decreasing byproducts, which proved to be challenging. Here, we have developed a human exonuclease 1-based genome-editing tool, referred to as exonuclease editor. When compared to Cas9, the exonuclease editor gave rise to increased HDR efficiency, reduced NHEJ repair frequency, and significantly elevated HDR/indel ratio. Robust gene editing precision of exonuclease editor was even superior to the fusion of Cas9 with E1B or DN1S, two previously reported precision-enhancing domains. Notably, exonuclease editor inhibited NHEJ at double strand breaks locally rather than globally, reducing indel frequency without compromising genome integrity. The replacement of Cas9 with single-strand DNA break-creating Cas9 nickase further increased the HDR/indel ratio by 453-fold than the original Cas9. In addition, exonuclease editor resulted in high microhomology-mediated end joining efficiency, allowing accurate and flexible deletion of targeted sequences with extended lengths with the aid of paired sgRNAs. Exonuclease editor was further used for correction of DMD patient-derived induced pluripotent stem cells, where 30.0% of colonies were repaired by HDR versus 11.1% in the control. Therefore, the exonuclease editor system provides a versatile and safe genome editing tool with high precision and holds promise for therapeutic gene correction.
Sections du résumé
BACKGROUND
BACKGROUND
Many efforts have been made to improve the precision of Cas9-mediated gene editing through increasing knock-in efficiency and decreasing byproducts, which proved to be challenging.
RESULTS
RESULTS
Here, we have developed a human exonuclease 1-based genome-editing tool, referred to as exonuclease editor. When compared to Cas9, the exonuclease editor gave rise to increased HDR efficiency, reduced NHEJ repair frequency, and significantly elevated HDR/indel ratio. Robust gene editing precision of exonuclease editor was even superior to the fusion of Cas9 with E1B or DN1S, two previously reported precision-enhancing domains. Notably, exonuclease editor inhibited NHEJ at double strand breaks locally rather than globally, reducing indel frequency without compromising genome integrity. The replacement of Cas9 with single-strand DNA break-creating Cas9 nickase further increased the HDR/indel ratio by 453-fold than the original Cas9. In addition, exonuclease editor resulted in high microhomology-mediated end joining efficiency, allowing accurate and flexible deletion of targeted sequences with extended lengths with the aid of paired sgRNAs. Exonuclease editor was further used for correction of DMD patient-derived induced pluripotent stem cells, where 30.0% of colonies were repaired by HDR versus 11.1% in the control.
CONCLUSIONS
CONCLUSIONS
Therefore, the exonuclease editor system provides a versatile and safe genome editing tool with high precision and holds promise for therapeutic gene correction.
Identifiants
pubmed: 38769511
doi: 10.1186/s12915-024-01918-w
pii: 10.1186/s12915-024-01918-w
doi:
Substances chimiques
EXO1 protein, human
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
119Subventions
Organisme : National Key Research and Development Program of China
ID : 2023YFF0724703
Organisme : National Natural Science Foundation of China
ID : 32200418
Organisme : Major Science and Technology Project of Hainan Province
ID : ZDKJ2021030
Organisme : Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences
ID : 2019-I2M-5-025
Organisme : China Postdoctoral Science Foundation
ID : 2022M723170
Organisme : Science and Technology Planning Project of Guangdong Province, China
ID : 2023B1212060050
Organisme : Science and Technology Planning Project of Guangdong Province, China
ID : 2021B1212040016
Organisme : Science and Technology Planning Project of Guangdong Province, China
ID : 2023A1515011312
Organisme : Science and Technology Planning Project of Guangdong Province, China
ID : 2021A1515110838
Organisme : Science and Technology Program of Guangzhou, China
ID : 202201010621
Informations de copyright
© 2024. The Author(s).
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