Precise CRISPR-Cas-mediated gene repair with minimal off-target and unintended on-target mutations in human hematopoietic stem cells
Open accessAbstract
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.
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CRISPR/Cas9 is more favored than ZFNs and TALENs and has revolutionized the biotechnology field.
“Designing sgRNA is more convenient, programmable and cost-effective than designing DNA binding domains, thus CRISPR/Cas9 is more favored than ZFNs and TALENs and has revolutionized the biotechnology field (Zhang et al., 2021; Tran et al., 2022).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Tran et al., 2022
The Introduction (page unavailable) calls CRISPR-Cas9 a powerful gene-editing tool, and Materials and Methods describes designing sgRNAs. Neither passage compares it with ZFNs or TALENs or says it is more favored or has revolutionized biotechnology. Searches for those comparisons and claims found no supporting passage in our copy. (LLM, conf=0.72)
Experimental — accuracy not yet measuredDesigning sgRNA is more convenient, programmable and cost-effective than designing DNA binding domains.
“Designing sgRNA is more convenient, programmable and cost-effective than designing DNA binding domains, thus CRISPR/Cas9 is more favored than ZFNs and TALENs and has revolutionized the biotechnology field (Zhang et al., 2021; Tran et al., 2022).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Tran et al., 2022
The Materials and Methods section describes designing and purchasing sgRNAs, but does not compare their design with DNA-binding domains. Searches for ZFNs, TALENs, and the claimed advantages found no passage in our copy addressing that comparison; page numbers were not provided. (LLM, conf=0.76)
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Off-target effects in CRISPR/Cas9 gene editing
Guo, C., Ma, X., Gao, F., & Guo, Y. (2023) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2023.1143157 · Cited by 625 · see citing works
“Designing sgRNA is more convenient, programmable and cost-effective than designing DNA binding domains, thus CRISPR/Cas9 is more favored than ZFNs and TALENs and has revolutionized the biotechnology field (Zhang et al., 2021; Tran et al., 2022).”
BackgroundGlaser, V., Flugel, C., Kath, J., Du, W., Drosdek, V., Franke, C., Stein, M., Pruß, A., Schmueck-Henneresse, M., Volk, H.-D., et al. (2023) · Genome biology · doi:10.1186/s13059-023-02928-7 · Cited by 65 · see citing works
- Authors
- Tran, N. T., Danner, E., Li, X., Graf, R., Lebedin, M., de la Rosa, K., Kühn, R., Rajewsky, K., & Chu, V. T.
- Affiliations
- Max Delbrück Center (DE)
- Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Immune Mechanisms and Human Antibodies, Berlin, Germany.
- Corresponding author
- Tran, Ngoc Tung
- Danner, Eric
- Li, Xun
- Graf, Robin
- Rajewsky, Klaus
- Chu, Van Trung
- ORCID
- Venue
- Science Advances
- Published
- 2022-06-03
- Volume
- 8
- Issue
- 22
- Pages
- eabm9106-eabm9106
- ISSN
- 2375-2548 (ISSN-L)
- PMC
- PMC9166625
- OpenAlex
- W4281741939
- Cited by
- 49
- References
- 50
- Citations by year
- 2026: 8 · 2025: 8 · 2024: 15 · 2023: 17 · 2022: 1
- FWCI
- 3.51
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- cc-by
- Locations
- Science Advances (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- MDC Repository (Max-Delbrueck-Center for Molecular Medicine) (submittedVersion)
- Funders
- National Heart, Lung, and Blood Institute (R21HL168669)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Health Sciences › Medicine › Oncology › CAR-T cell therapy research
- Keywords
- CRISPR/Cas9, Cas9 nickase, off-target mutations, hematopoietic stem cells, hematopoietic stem and progenitor cells, homology-directed repair, non-homologous end joining, gene therapy safety, sickle cell disease, beta-globin, neutrophil elastase, CD127, PRF1, T cells
- MeSH terms
- Gene Editing, Dependovirus, Hematopoietic Stem Cells, Humans, Mutation, Genetic Therapy, CRISPR-Cas Systems
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