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| 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., Lebedin, M., Danner, E., Kühn, R., 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) |
| Source: | Bio-protocol |
| ISSN: | 2331-8325 |
| Publisher: | Bio-protocol LLC |
| Volume: | 13 |
| Number: | 8 |
| Page Range: | e4661 |
| Date: | 20 April 2023 |
| Official Publication: | https://doi.org/10.21769/BioProtoc.4661 |
| PubMed: | View item in PubMed |
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