Helmholtz Gemeinschaft


Application of a spacer-nick gene-targeting approach to repair disease-causing mutations with increased safety

PDF (Original Article) - Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader

Item Type:Article
Title:Application of a spacer-nick gene-targeting approach to repair disease-causing mutations with increased safety
Creators Name:Tran, N.T. and Lebedin, M. and Danner, E. and Kühn, R. and Rajewsky, K. and Chu, V.T.
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 spacer-nick gene repair approach that combines Cas9(D10A) 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.
Keywords:Cas9D10A Nickase, PAM-Out sgRNAs, Spacer Distance of 200-350 bp, Gene Repair Approach, AAV6, Gene Correction Efficiency, Unintended On-Target Mutations, Off-Target Mutations, Human Hematopoietic Stem and Progenitor Cells (HSPCs)
Publisher:Bio-protocol LLC
Page Range:e4661
Date:20 April 2023
Official Publication:https://doi.org/10.21769/BioProtoc.4661
PubMed:View item in PubMed

Repository Staff Only: item control page


Downloads per month over past year

Open Access
MDC Library