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Precise CRISPR/Cas-mediated gene repair with minimal off-target and unintended on-target mutations in human hematopoietic stem cells

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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. and Danner, E. and Li, X. and Graf, R. and Lebedin, M. and de la Rosa, K. and Kühn, R. and 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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