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Application of a Spacer-nick Gene-targeting Approach to Repair Disease-causing Mutations with Increased Safety

Overview
Journal Bio Protoc
Specialty Biology
Date 2023 Apr 28
PMID 37113334
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Abstract

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 gene repair approach that combines Cas9 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.

Citing Articles

Expansion and Precise CRISPR-Cas9 Gene Repair of Autologous T-Memory Stem Cells from Patients with T-Cell Immunodeficiencies.

Li X, Chu V, Kocks C, Rajewsky K Bio Protoc. 2024; 14(20):e5085.

PMID: 39512884 PMC: 11540044. DOI: 10.21769/BioProtoc.5085.

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