| Autor: |
Ngoc Tung Tran, Mikhail Lebedin, Eric Danner, Ralf Kühn, Klaus Rajewsky, Van Trung Chu |
| Jazyk: |
angličtina |
| Rok vydání: |
2023 |
| Předmět: |
|
| Zdroj: |
Bio-Protocol, Vol 13, Iss 8 (2023) |
| Druh dokumentu: |
article |
| ISSN: |
2331-8325 |
| DOI: |
10.21769/BioProtoc.4661 |
| Popis: |
The CRISPR/Cas9 system is a powerful tool for gene repair that holds great potential for gene therapy to cure monogenic diseases. Despite intensive improvement, the safety of this system remains a major clinical concern. In contrast to Cas9 nuclease, Cas9 nickases with a pair of short-distance (38–68 bp) PAM-out single-guide RNAs (sgRNAs) preserve gene repair efficiency while strongly reducing off-target effects. However, this approach still leads to efficient unwanted on-target mutations that may cause tumorigenesis or abnormal hematopoiesis. We establish a precise and safe spacer-nick gene repair approach that combines Cas9D10A nickase with a pair of PAM-out sgRNAs at a distance of 200–350 bp. In combination with adeno-associated virus (AAV) serotype 6 donor templates, this approach leads to efficient gene repair with minimal unintended on- and off-target mutations in human hematopoietic stem and progenitor cells (HSPCs). Here, we provide detailed protocols to use the spacer-nick approach for gene repair and to assess the safety of this system in human HSPCs. The spacer-nick approach enables efficient gene correction for repair of disease-causing mutations with increased safety and suitability for gene therapy.Graphical overview |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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