ArticleGenome biology2023
Combining different CRISPR nucleases for simultaneous knock-in and base editing prevents translocations in multiplex-edited CAR T cells.
Article in Genome biology, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. An erratum has been issued. Cited by 50 papers.
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Who cites it
50 citing papers in PubMed, 57 citations in OpenAlex.
- Human T cell engineering via serial delivery of mRNA encapsulated within lipid nanoparticles.Molecular therapy. Nucleic acids · 2026Article
- Review
- CRISPR-Engineered CAR-T Cell Therapy for Epstein-Barr Virus-Associated Nasopharyngeal Carcinoma: A Review of Emerging Therapeutic Prospects.Reviews in medical virology · 2026Review
- CRISPR integration of an FLT3 CAR into the TRAC locus generates CAR T cells for AML.Genome medicine · 2026Article
- Adenine base editor for knockout of proteins: A practical guide from design to analysis with updated MultiEditRbatch.Molecular therapy. Nucleic acids · 2026Article
- Precise, predictable genome integrations by deep-learning-assisted design of microhomology-based templates.Nature biotechnology · 2026Article
- Simultaneous orthogonal cell engineering by a single CRISPR-Cas9 polyfunctional editor.Nature communications · 2026Article
- Strategies to eliminate native T cell receptors for adoptive T cell therapies.Molecular therapy : the journal of the American Society of Gene Therapy · 2026Review
- From Bench to Bedside: Ethical and Clinical Best Practices for Genome Editing Applications.International journal of molecular sciences · 2026Review
- Engineering CAR-T cells for solid tumors: Overcoming the microenvironment through integrated design and clinical translation.Oncoscience · 2026Review
- CRISPR/methotrexate-integrated strategy for TCR-T cell engineering with reduced chromosome 14 loss.Molecular therapy. Methods & clinical development · 2025Article
- Orthogonal CRISPR systems for targeted integration and multiplex base editing enable nonviral engineering of allogeneic CAR-T cells.Molecular therapy : the journal of the American Society of Gene Therapy · 2025Article
- Viral vector-free generation of orthogonal IL-2-responsive CAR T cells through gene editing of IL-2 and its receptor.Blood immunology & cellular therapy · 2025Article
- Next-generation T cell immunotherapies engineered with CRISPR base and prime editing: challenges and opportunities.Nature reviews. Clinical oncology · 2025Review
- Integration of humanized ROBO1 CAR in PD-1 locus in natural killer cells delivers synergistic tumor-killing effect against non-small cell lung cancer.Cancer gene therapy · 2025Article
- HLA matching or CRISPR editing of HLA class I/II enables engraftment and effective function of allogeneic human regulatory T cell therapy in a humanized mouse transplantation model.Nature communications · 2025Article
- Base-editing a single missense mutation in A20 enhances CAR-T cell efficacy.bioRxiv : the preprint server for biology · 2025Article
- Fine tuning towards the next generation of engineered T cells.Nature biomedical engineering · 2025Review
- CRISPR tools for T cells: targeting the genome, epigenome, and transcriptome.Trends in cancer · 2025Review
- Engineering CAR-T cells for solid tumors: bispecific antigen targeting, tumor microenvironment modulation, and toxicity control.Immunologic research · 2025Review
Corrections and comments
- Erratum issued
Authors and funding
12 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundMultiple genetic modifications may be required to develop potent off-the-shelf chimeric antigen receptor (CAR) T cell therapies. Conventional CRISPR-Cas nucleases install sequence-specific DNA double-strand breaks (DSBs), enabling gene knock-out or targeted transgene knock-in. However, simultaneous DSBs provoke a high rate of genomic rearrangements which may impede the safety of the edited cells.
resultsHere, we combine a non-viral CRISPR-Cas9 nuclease-assisted knock-in and Cas9-derived base editing technology for DSB free knock-outs within a single intervention. We demonstrate efficient insertion of a CAR into the T cell receptor alpha constant (TRAC) gene, along with two knock-outs that silence major histocompatibility complexes (MHC) class I and II expression. This approach reduces translocations to 1.4% of edited cells. Small insertions and deletions at the base editing target sites indicate guide RNA exchange between the editors. This is overcome by using CRISPR enzymes of distinct evolutionary origins. Combining Cas12a Ultra for CAR knock-in and a Cas9-derived base editor enables the efficient generation of triple-edited CAR T cells with a translocation frequency comparable to unedited T cells. Resulting TCR- and MHC-negative CAR T cells resist allogeneic T cell targeting in vitro.
conclusionsWe outline a solution for non-viral CAR gene transfer and efficient gene silencing using different CRISPR enzymes for knock-in and base editing to prevent translocations. This single-step procedure may enable safer multiplex-edited cell products and demonstrates a path towards off-the-shelf CAR therapeutics.
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