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. 2019 Nov 6;9:16104. doi: 10.1038/s41598-019-52616-5

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© The Author(s) 2019

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Identification of a tracrRNA with improved Cas9 RNP activity. (A) A schematic of the crRNA and tracrRNA. The target sequence is represented by N(20) in the crRNA. The boxed nucleotides are altered in tracrRNA-6 and 19. (B) A series of U-modified tracrRNAs (1–17) were annealed with a TAR6 crRNA and transfected into pMoHIV-C6 cells. GFP expression was assessed by FACS at 48 hours post-transfection. An unmodified tracrRNA-(tracrRNA-UM) was included as a comparative control. Untransfected cells (Mock) or a transfection without a dgRNA (control) were included as negative controls. (C) A serial dilution of the tracrRNA-6 and tracrRNA-UM were transfected into pMoHIV-C6 cells, and the levels of GFP were assessed by FACS. The embedded image reflects fold change in knock-down activity relative to the tracrRNA-UM at each dilution. (D) Total DNA was extracted 48 hours post-transfection and the percentage of indels was determined by drop-off assay using ddPCR. The embedded image reflects fold change in indels relative to the tracrRNA-UM at each dilution of the tracrRNA-6. The errors bars represent standard error of the mean (SEM) of duplicate treated samples and experiments performed in duplicate, and the experiment was repeated twice.*p < 0.05, ***p < 0.001 were obtained by one-way ANOVA and Dunnett’s test.