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. 2021 Jun 3;11:11818. doi: 10.1038/s41598-021-91307-y

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Persistent knockout of endogenous TCR in primary human T cells with Cas9 and µVS. Cas9 and locus-specific TRAC-1 gRNAs (as Cas9–RNP complexes) were delivered to activated primary human CD3⁺ T cells via µVS or electroporation. Quantification of (A) percentage of TCR knockout cells, (B) total viable, TCR knockout cells, (C) percentage of viable T cells and (D) total viable T cells were performed at Days 1, 4, 7, 10 and 14 post-transfection. TCR expression was measured via CD3 and TCRα/β co-staining. TCR knockout was quantified as a percentage of CD3- and TCRα/β-double negative cells and the total number of live cells. Propidium iodide exclusion gating and event collection rate were used to measure viability and cell concentration, respectively. Non-targeting gRNA as a Cas9–RNP complex served as a non-editing control. Non-transfected samples served as a negative control. Data represents mean ± SD of n = 3 samples per condition and ≥ 2 independent experiments. *P < 0.05, **P < 0.01 by unpaired, two-tailed, heteroscedastic t tests. Figure generated using Adobe Illustrator Creative Cloud (adobe.com/products/illustrator.html) and GraphPad Prism 9 (graphpad.com/scientific-software/prism/).