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. 2021 Nov 1;12:6267. doi: 10.1038/s41467-021-26518-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 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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Fig. 2 — a In vivo experimental plan to validate rAAV:Nme2Cas9 Dual-sgRNA:Designs 1 and 4 to disrupt the Hpd gene by tail vein injections of AAV8 vectors in adult Fah^neo/neo and C57BL/6 mice. b Complete rescue of body weights after Dual-sgRNA:Designs 1 and 4 in Fah^neo/neo mice. c Quantification of editing events in C57BL/6 (left) and Fah^neo/neo (right) mice by SMRT sequencing analysis showing efficiencies of segmental deletion (orange) and inversion (blue) outcomes. d Bar graph showing the percentages of indels recorded in full-length (3.6 kb), UMI-corrected SMRT reads after Hpd editing by AAV8 delivery of Dual-sgRNA:Designs 1 and 4 in C57BL/6 (left) and Fah^neo/neo (right) mice. The plot indicates indels recorded only at gRNA-I (in red), indels only at gRNA-II (in dark green), and indels at both gRNA-I and gRNA-II (yellow) as measured by SMRT sequence analysis. e rAAV fragment integration as detected by SMRT sequencing analysis. f Quantitation of editing events in UMI-corrected UDiTaS analysis reads after Hpd editing by AAV8 delivery of Dual-sgRNA:Design 4 in C57BL/6 and Fah^neo/neo mice showing mean efficiencies of segmental deletion, inversion, and AAV fragment integration. g mRNA levels from RNA-seq (transcripts per million) of Hpd wild-type mRNA in the livers of C57BL/6 mice and Fah^neo/neo mice. h Volcano plots showing differentially expressed genes with false discovery rate ≤5% after adjusting for multiple comparisons (red; analysis detailed in Methods) between Dual-sgRNA:Design 1 and PBS-treated Fah^neo/neo mice (left) and Dual-sgRNA:Design 4 and PBS-treated Fah^neo/neo mice (right). i Total HPD protein knockout as shown by anti-HPD Western blot using total protein collected from mouse liver homogenates. This analysis of samples from multiple mice was performed once. j Representative images of immunostaining for HPD in liver tissues in Fah^neo/neo mice injected with PBS (left) or Dual-sgRNA:Design 4 (right). Scale bar is 100 μm. All mice in the cohort were examined once by this method (see Supplementary Fig. 2). Data were presented as mean values ± s.e.m. Sample size in panels b–g: (n = 3 PBS-injected C57BL/6 mice; n = 4 Dual-sgRNA.Design 1 or 4 injected C57BL/6 mice; n = 1 in pre-NTBC Fah^neo/neo cohort; n = 7 in post-NTBC Dual-sgRNA.Design 1 or 4 injected Fah^neo/neo cohort).