ArticleMolecular therapy : the journal of the American Society of Gene Therapy2022
Prediction and validation of hematopoietic stem and progenitor cell off-target editing in transplanted rhesus macaques.
Article in Molecular therapy : the journal of the American Society of Gene Therapy, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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Who cites it
13 citing papers in PubMed, 20 citations in OpenAlex.
- Variant-aware Cas-OFFinder: web-based in silico variant-aware potential off-target site identification for genome editing applications.Nucleic acids research · 2025Article
- Advancing CRISPR genome editing into gene therapy clinical trials: progress and future prospects.Expert reviews in molecular medicine · 2025Review
- Impact of CRISPR/HDR editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSPCs in rhesus macaques.Cell stem cell · 2024Article
- Clonal hematopoiesis in people with advanced HIV and associated inflammatory syndromes.JCI insight · 2024Article
- Efficient long-term multilineage engraftment of CD33-edited hematopoietic stem/progenitor cells in nonhuman primates.Molecular therapy. Methods & clinical development · 2023Article
- Challenges of CRISPR/Cas-Based Cell Therapy for Type 1 Diabetes: How Not to Engineer a "Trojan Horse".International journal of molecular sciences · 2023Review
- Gene therapy for sickle cell disease.Hematology. American Society of Hematology. Education Program · 2023Article
- Article
- Article
- T-CAST: An optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds.Frontiers in genome editing · 2023Article
- Genome editing for primary immunodeficiencies: A therapeutic perspective on Wiskott-Aldrich syndrome.Frontiers in immunology · 2022Review
- Understanding and overcoming adverse consequences of genome editing on hematopoietic stem and progenitor cells.Molecular therapy : the journal of the American Society of Gene Therapy · 2021Review
- Genome editing in large animal models.Molecular therapy : the journal of the American Society of Gene Therapy · 2021Review
Corrections and comments
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Authors and funding
23 authors at 10 institutions in 3 countries.
Funding
Abstract
The programmable nuclease technology CRISPR-Cas9 has revolutionized gene editing in the last decade. Due to the risk of off-target editing, accurate and sensitive methods for off-target characterization are crucial prior to applying CRISPR-Cas9 therapeutically. Here, we utilized a rhesus macaque model to compare the predictive values of CIRCLE-seq, an in vitro off-target prediction method, with in silico prediction (ISP) based solely on genomic sequence comparisons. We use AmpliSeq HD error-corrected sequencing to validate off-target sites predicted by CIRCLE-seq and ISP for a CD33 guide RNA (gRNA) with thousands of off-target sites predicted by ISP and CIRCLE-seq. We found poor correlation between the sites predicted by the two methods. When almost 500 sites predicted by each method were analyzed by error-corrected sequencing of hematopoietic cells following transplantation, 19 off-target sites revealed insertion or deletion mutations. Of these sites, 8 were predicted by both methods, 8 by CIRCLE-seq only, and 3 by ISP only. The levels of cells with these off-target edits exhibited no expansion or abnormal behavior in vivo in animals followed for up to 2 years. In addition, we utilized an unbiased method termed CAST-seq to search for translocations between the on-target site and off-target sites present in animals following transplantation, detecting one specific translocation that persisted in blood cells for at least 1 year following transplantation. In conclusion, neither CIRCLE-seq or ISP predicted all sites, and a combination of careful gRNA design, followed by screening for predicted off-target sites in target cells by multiple methods, may be required for optimizing safety of clinical development.
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