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| Item Type: | Article |
|---|---|
| Title: | Precise CRISPR/Cas-mediated gene repair with minimal off-target and unintended on-target mutations in human hematopoietic stem cells |
| Creators Name: | Tran, N.T., Danner, E., Li, X., Graf, R., Lebedin, M., de la Rosa, K., Kühn, R., Rajewsky, K. and Chu, V.T. |
| Abstract: | While CRISPR-Cas9 is key for the development of gene therapy, its potential off-target mutations are still a major concern. Here, we establish a "spacer-nick" gene correction approach that combines the Cas9(D10A) nickase with a pair of PAM-out sgRNAs at a distance of 200 to 350 bp. In combination with adeno-associated virus (AAV) serotype 6 template delivery, our approach led to efficient HDR in human hematopoietic stem and progenitor cells (HSPCs including long-term HSCs) and T cells, with minimal NHEJ-mediated on-target mutations. Using spacer-nick, we developed an approach to repair disease-causing mutations occurring in the HBB, ELANE, IL7R, and PRF1 genes. We achieved gene correction efficiencies of 20 to 50% with minimal NHEJ-mediated on-target mutations. On the basis of in-depth off-target assessment, frequent unintended genetic alterations induced by classical CRISPR-Cas9 were significantly reduced or absent in the HSPCs treated with spacer-nick. Thus, the spacer-nick gene correction approach provides improved safety and suitability for gene therapy. |
| Keywords: | CRISPR-Cas Systems, Dependovirus, Gene Editing, Genetic Therapy, Hematopoietic Stem Cells, Mutation |
| Source: | Science Advances |
| ISSN: | 2375-2548 |
| Publisher: | American Association for the Advancement of Science |
| Volume: | 8 |
| Number: | 22 |
| Page Range: | eabm9106 |
| Date: | 3 June 2022 |
| Official Publication: | https://doi.org/10.1126/sciadv.abm9106 |
| PubMed: | View item in PubMed |
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