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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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Surface marker expression and cytokine secretion levels minimally perturbed with Cas9 and µVS. (A) CD25, and (B) PD-1 expression levels were quantified in cells transfected with µVS or electroporation. Surface marker expression levels were measured in the total live cell and TRAC-1 KO live population via flow cytometry at Days 1, 4, 7, 10 and 14 post-transfection. (C) ELISA quantification of IFNγ supernatant levels in transfected and non-transfected cells at Day 1, 4, 7, and 10 post-transfection. Non-targeting gRNA as a Cas9–RNP complex served as a non-editing control. Cells mixed with P3 buffer (EP—non-transfection) or Opti-MEM buffer (µVS—non-transfection) that were not transfected served as non-transfection controls for electroporation and µVS samples, respectively. Data represent mean ± SD of n = 3 samples per condition. *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/).