학술논문
Genome-wide off-target analyses of CRISPR/Cas9-mediated T-cell receptor engineering in primary human T cells.
Document Type
Academic Journal
Author
Kaeuferle T; Department of Pediatric Hematology, Oncology, Hemostaseology and Stem Cell Transplantation Dr. von Hauner Children's Hospital University Hospital, LMU Munich Munich Germany.; German Center for Infection Research (DZIF) Munich Germany.; Stief TA; Department of Pediatric Hematology, Oncology, Hemostaseology and Stem Cell Transplantation Dr. von Hauner Children's Hospital University Hospital, LMU Munich Munich Germany.; German Center for Infection Research (DZIF) Munich Germany.; Canzar S; Gene Center Ludwig Maximilians University of Munich Munich Germany.; Kutlu NN; Department of Pediatric Hematology, Oncology, Hemostaseology and Stem Cell Transplantation Dr. von Hauner Children's Hospital University Hospital, LMU Munich Munich Germany.; Willier S; Department of Pediatric Hematology, Oncology, Hemostaseology and Stem Cell Transplantation Dr. von Hauner Children's Hospital University Hospital, LMU Munich Munich Germany.; Stenger D; Department of Pediatric Hematology, Oncology, Hemostaseology and Stem Cell Transplantation Dr. von Hauner Children's Hospital University Hospital, LMU Munich Munich Germany.; Ferrada-Ernst P; Department of Pediatric Hematology, Oncology, Hemostaseology and Stem Cell Transplantation Dr. von Hauner Children's Hospital University Hospital, LMU Munich Munich Germany.; Habjan N; Department of Pediatric Hematology, Oncology, Hemostaseology and Stem Cell Transplantation Dr. von Hauner Children's Hospital University Hospital, LMU Munich Munich Germany.; Peters AE; Department of Pediatric Hematology, Oncology, Hemostaseology and Stem Cell Transplantation Dr. von Hauner Children's Hospital University Hospital, LMU Munich Munich Germany.; Busch DH; German Center for Infection Research (DZIF) Munich Germany.; Institute for Medical Microbiology, Immunology and Hygiene Technische Universität München (TUM) Munich Germany.; Feuchtinger T; Department of Pediatric Hematology, Oncology, Hemostaseology and Stem Cell Transplantation Dr. von Hauner Children's Hospital University Hospital, LMU Munich Munich Germany.; German Center for Infection Research (DZIF) Munich Germany.
Source
Publisher: John Wiley & Sons Australia, Ltd. on behalf of Australasian Society for Immunology Inc Country of Publication: Australia NLM ID: 101638268 Publication Model: eCollection Cited Medium: Print ISSN: 2050-0068 (Print) Linking ISSN: 20500068 NLM ISO Abbreviation: Clin Transl Immunology Subsets: PubMed not MEDLINE
Subject
Language
English
ISSN
2050-0068
Abstract
Objectives: Exploiting the forces of human T cells for treatment has led to the current paradigm of emerging immunotherapy strategies. Genetic engineering of the T-cell receptor (TCR) redirects specificity, ablates alloreactivity and brings significant progress and off-the-shelf options to emerging adoptive T-cell transfer (ACT) approaches. Targeted CRISPR/Cas9-mediated double-strand breaks in the DNA enable knockout or knock-in engineering.
Methods: Here, we perform CRISPR/Cas9-mediated TCR knockout using a therapeutically relevant ribonucleoprotein (RNP) delivery method to assess the safety of genetically engineered T-cell products. Whole-genome sequencing was performed to analyse whether CRISPR/Cas9-mediated DNA double-strand break at the TCR locus is associated with off-target events in human primary T cells.
Results: TCRα chain and TCRβ chain knockout leads to high on-target InDel frequency and functional knockout. None of the predicted off-target sites could be confirmed experimentally, whereas whole-genome sequencing and manual Integrative Genomics Viewer (IGV) review revealed 9 potential low-frequency off-target events genome-wide. Subsequent amplification and targeted deep sequencing in 7 of 7 evaluable loci did not confirm these low-frequency InDels. Therefore, off-target events are unlikely to be caused by the CRISPR/Cas9 engineering.
Conclusion: The combinatorial approach of whole-genome sequencing and targeted deep sequencing confirmed highly specific genetic engineering using CRISPR/Cas9-mediated TCR knockout without potentially harmful exonic off-target effects.
Competing Interests: The authors have no conflict of interest to disclose.
(© 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)
Methods: Here, we perform CRISPR/Cas9-mediated TCR knockout using a therapeutically relevant ribonucleoprotein (RNP) delivery method to assess the safety of genetically engineered T-cell products. Whole-genome sequencing was performed to analyse whether CRISPR/Cas9-mediated DNA double-strand break at the TCR locus is associated with off-target events in human primary T cells.
Results: TCRα chain and TCRβ chain knockout leads to high on-target InDel frequency and functional knockout. None of the predicted off-target sites could be confirmed experimentally, whereas whole-genome sequencing and manual Integrative Genomics Viewer (IGV) review revealed 9 potential low-frequency off-target events genome-wide. Subsequent amplification and targeted deep sequencing in 7 of 7 evaluable loci did not confirm these low-frequency InDels. Therefore, off-target events are unlikely to be caused by the CRISPR/Cas9 engineering.
Conclusion: The combinatorial approach of whole-genome sequencing and targeted deep sequencing confirmed highly specific genetic engineering using CRISPR/Cas9-mediated TCR knockout without potentially harmful exonic off-target effects.
Competing Interests: The authors have no conflict of interest to disclose.
(© 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)