Erratum: CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy.

Abstract

An erratum was issued for: Studying TGF-β Signaling and TGF-β-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells. The phrases\xa0 surveyor assay \xa0and\xa0 Surveyor\xa0Nuclease have\xa0been updated to T7E1 assay \xa0 to T7\xa0endonuclease I respectively.\xa0 Step 1.2 in\xa0the Protocol has been updated from: Surveyor nuclease\xa0assay of sgRNA NOTE: The targeting efficiency of the sgRNA used for the knock-in experiment is evaluated by surveyor nuclease assay (also known as T7 endonuclease I (T7EI) assay)17. Select the sgRNA with high DNA cleavage efficiency and a low distance between the sgRNA cutting site and the stop codon. to: T7 endonuclease assay of sgRNA NOTE: The targeting efficiency of the sgRNA used for the knock-in experiment is evaluated by T7 endonuclease (T7EI)\xa0assay17. Select the sgRNA with high DNA cleavage efficiency and a low distance between the sgRNA cutting site and the stop codon. Figure 1 in the Representative Results has been updated\xa0from: Figure 1: HMEJ-mediated targeted integration in vitro. (A) Experimental scheme for selection of sgRNAs: Six different sgRNAs (Cdx2-sgRNA1~Cdx2-sgRNA6) around the stop codon of the Cdx2 locus with a higher rank and off-target potential were chosen based on online CRISPR design tool. The protospacer adjacent motif (PAM) sequence is in red. (B) Experimental design: The Cas9-CMV-GFP expression plasmids expressing sgRNA, Cas9, and GFP were introduced into N2a\xa0cells. GFP+ cells were sorted at day 3 for surveyor assay. (C) Surveyor assay for Cdx2 targeting: 6 different sgRNAs were designed for surveyor assay. Normal N2a cell genomic DNA serves as control. *, the sgRNA used for Cdx2-2A-mCherry knock-in experiment. (D) Schematic overview of construction of HMEJ donors using Gibson assembly. (E) Schematic overview of HMEJ-mediated gene targeting strategy at Cdx2 locus. HAL/HAR, left/right homology arm; triangles, sgRNA target sites; OF/OR, outer forward/reverse primer; IF/IR, inner forward/reverse primer. Figure modified from previous report10.\xa0Please click here to view a larger version of this figure. to: Figure 1: HMEJ-mediated targeted integration in vitro. (A) Experimental scheme for selection of sgRNAs: Six different sgRNAs (Cdx2-sgRNA1~Cdx2-sgRNA6) around the stop codon of the Cdx2 locus with a higher rank and off-target potential were chosen based on online CRISPR design tool. The protospacer adjacent motif (PAM) sequence is in red. (B) Experimental design: The Cas9-CMV-GFP expression plasmids expressing sgRNA, Cas9, and GFP were introduced into N2a\xa0cells. GFP+ cells were sorted at day 3 for T7EI\xa0assay. (C) T7EI assay for Cdx2 targeting: 6 different sgRNAs were designed for T7EI assay. Normal N2a cell genomic DNA serves as control. *, the sgRNA used for Cdx2-2A-mCherry knock-in experiment. (D) Schematic overview of construction of HMEJ donors using Gibson assembly. (E) Schematic overview of HMEJ-mediated gene targeting strategy at Cdx2 locus. HAL/HAR, left/right homology arm; triangles, sgRNA target sites; OF/OR, outer forward/reverse primer; IF/IR, inner forward/reverse primer. Figure modified from previous report10.\xa0Please click here to view a larger version of this figure.