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

Kaeuferle, Theresa; Stief, Tanja A; Canzar, Stefan; Kutlu, Nayad N; Willier, Semjon; Stenger, Dana; Ferrada‐Ernst, Paulina; Habjan, Nicola; Peters, Annika E; Busch, Dirk H; Feuchtinger, Tobias (2022): Genome‐wide off‐target analyses of CRISPR/Cas9‐mediated T‐cell receptor engineering in primary human T cells. Clinical & Translational Immunology, 11 (1). 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.

• Doc-Type: Article (LMU Hospital)
• Organisational unit (Faculties): 07 Medicine > Medical Center of the University of Munich > Pediatric Clinic and Outpatient Clinic in the Dr. von Hauner Children‘s Hospital
• DFG subject classification of scientific disciplines: Life sciences
• DFG: Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - 491502892