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. 2023 Aug 17;14:4998. doi: 10.1038/s41467-023-40701-3

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© The Author(s) 2023

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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Fig. 5 — a Schematic overview of the problem arising when CBE and ABE is applied using the same cas9-homolog. This can be circumvented by using Sa-CBE together with Sp-ABE. b Brightfield images showing hepatocyte organoid outgrowth upon Nutlin-3 selection on 3 SaKKH-sgRNA’s compared to a scrambled control sgRNA and our optimal TP53^W53* sgRNA in combination with SpCas9-CBE. Scale bars are 2000 µm. c Sanger sequencing of hepatocyte organoids surviving Nutlin-3 selection upon transfection of SaKKH-CBE and a TP53^W146* sgRNA. Edited residue is highlighted with an asterisk and SaKKH PAM is shown in yellow. d Quantification of editing observed in Nutlin-3 resistant clones highlights increased indel induction by SaKKH-CBE. Clones with homozygous C > T mutations are shown in green and clones with indels on one of their alleles are shown in orange. e Quantification of editing observed in Nutlin-3 resistant clones highlights increased amount of unintended edits upon SaKKH-CBE editing. Pie-chart shows clones with homozygous C > T (green) mutations and clones harboring a C > A (red), C > G (blue), or indel (orange) on at least one of their alleles. f Brightfield images of organoids upon Cas9-homolog multiplexing using SpCas9-ABE to target CTNNB1 and SaKKHcas9-CBE to target TP53. Organoids are selected for TP53 mutations by addition of Nutlin-3 to the culture media, and selected for CTNNB1 mutations by addition removal of CHIR and Rspondin-1. Scale bars are 2000 µm. g Brightfield images of Cas9-homolog multiplexing. Organoids are transfected with SaKKH-CBE targeting TP53 and APC and SpCas9-NG targeting PIK3CA^H1047R. Organoids are selected on medium lacking wnt and Rspondin-1 (rpso) and with the addition of Nutlin-3(Nut-3)Scale bars are 2000 µm. h Sanger sequencing of the PIK3CA^H1047R locus showing heterozygous mutation induction. PAM is shown in blue and mutation is highlighted with an asterisk. i Quantification of editing efficiency of the SpCas9-NG PIK3CA^H1047R sgRNA by Sanger sequencing. Hom homozygous, Het heterozygous, WT wild type. Source data are provided as a Source data file.