Genome-wide off-target analyses of CRISPR/Cas9-mediated T-cell receptor engineering in primary human T cells

Theresa Kaeuferle, Tanja A. Stief, Stefan Canzar, Nayad N. Kutlu, Semjon Willier, Dana Stenger, Paulina Ferrada-Ernst, Nicola Habjan, Annika E. Peters, Dirk H. Busch, Tobias Feuchtinger

Research output: Contribution to journalArticlepeer-review

5 Scopus citations


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.

Original languageEnglish (US)
Article numbere1372
JournalClinical and Translational Immunology
Issue number1
StatePublished - 2022

All Science Journal Classification (ASJC) codes

  • General Nursing
  • Immunology and Allergy
  • Immunology


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