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. 2022 Jan 8;50(2):1187–1197. doi: 10.1093/nar/gkab1295

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© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 1. — Generation and characterization of H9-iPE2. (A) Schematic diagram of the strategy for TALEN-mediated targeting of the AAVS1 locus to generate H9-iPE2 cells, in which PE2 expression is induced by dox. The AAVS1 donor vector contains a cassette in which PE2 expression is under the control of the dox-inducible TRE3G promoter. SA, splice acceptor; 2A, self-cleaving 2A peptide; Neo, neomycin resistance gene; rtTA, dox-controlled reverse transcriptional activator; CAG, cytomegalovirus early enhancer/chicken β actin promoter. (B) PCR-based confirmation that the iPE2 construct was targeted to the AAVS1 locus. A primer pair that flanks the AAVS1 knock-in site only amplifies a product in wild-type cells; lack of a product indicates that the H9-iPE2 clone used in this study is homozygous (left panel). Junction PCR confirmed on-target integration of the cassette into the AAVS1 locus (center and right panels). (C) Immunostaining of H9-iPE2 cells before and after 48 h of dox treatment with an anti-Cas9 antibody (green). Nuclei were stained with DAPI (blue). (D) Editing efficiency in H9-iPE2 cells. H9-iPE2 cells maintained in the presence of dox were electroporated with plasmids encoding a pegRNA and a nicking sgRNA (for PE3 system). The editing efficiency is indicated as the percentage of total sequencing reads that contain the intended edit and do not contain indels. Mean ± s.d. of n = 3 independent biological replicates.