J Vis Exp. 2021 Mar 10(169). doi: 10.3791/6459.
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 "surveyor assay" and "Surveyor Nuclease" have been updated to "T7E1 assay" to " T7 endonuclease I" respectively. Step 1.2 in the Protocol has been updated from: Surveyor nuclease assay 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). 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) assay. 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 from: Figure 1: HMEJ-mediated targeted integration in vitro. (A) Experimental scheme for selection of sgRNAs: Six different sgRNAs (Cdx2-sgRNA1Cdx2-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 cells. 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 report. Please 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-sgRNA1Cdx2-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 cells. GFP cells were sorted at day 3 for T7EI assay. (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 report. Please click here to view a larger version of this figure.
《研究TGF-β信号传导及TGF-β诱导的乳腺癌和正常细胞上皮-间质转化》。“Surveyor检测法”和“Surveyor核酸酶”分别更新为“T7E1检测法”和“T7核酸内切酶I”。方案中的步骤1.2已从：Surveyor核酸酶检测sgRNA 注意：用于敲入实验的sgRNA的靶向效率通过Surveyor核酸酶检测法（也称为T7核酸内切酶I（T7EI）检测法）进行评估。选择具有高DNA切割效率且sgRNA切割位点与终止密码子之间距离较短的sgRNA。更新为：T7核酸内切酶检测sgRNA 注意：用于敲入实验的sgRNA的靶向效率通过T7核酸内切酶（T7EI）检测法进行评估。选择具有高DNA切割效率且sgRNA切割位点与终止密码子之间距离较短的sgRNA。代表性结果中的图1已从：图1：体外HMEJ介导的靶向整合。（A）sgRNA选择的实验方案：根据在线CRISPR设计工具，在Cdx2基因座终止密码子周围选择六个不同的sgRNA（Cdx2-sgRNA1Cdx2-sgRNA6），其具有较高的排名和脱靶潜力。原间隔相邻基序（PAM）序列用红色表示。（B）实验设计：将表达sgRNA、Cas9和GFP的Cas9-CMV-GFP表达质粒导入N2a细胞。在第3天对GFP细胞进行分选以进行Surveyor检测。（C）Cdx2靶向的Surveyor检测：设计6种不同的sgRNA用于Surveyor检测。正常N2a细胞基因组DNA用作对照。*，用于Cdx2-2A-mCherry敲入实验的sgRNA。（D）使用Gibson组装构建HMEJ供体的示意图概述。（E）Cdx2基因座处HMEJ介导的基因靶向策略的示意图概述。HAL/HAR，左/右同源臂；三角形，sgRNA靶位点；OF/OR，外部正向/反向引物；IF/IR，内部正向/反向引物。图改编自先前的报告。请点击此处查看此图的更大版本。更新为：图1：体外HMEJ介导的靶向整合。（A）sgRNA选择的实验方案：根据在线CRISPR设计工具，在Cdx2基因座终止密码子周围选择六个不同的sgRNA（Cdx2-sgRNA1Cdx2-sgRNA6），其具有较高的排名和脱靶潜力。原间隔相邻基序（PAM）序列用红色表示。（B）实验设计：将表达sgRNA、Cas9和GFP的Cas9-CMV-GFP表达质粒导入N2a细胞。在第3天对GFP细胞进行分选以进行T7EI检测。（C）Cdx2靶向的T7EI检测：设计6种不同的sgRNA用于T7EI检测。正常N2a细胞基因组DNA用作对照。*，用于Cdx2-2A-mCherry敲入实验的sgRNA。（D）使用Gibson组装构建HMEJ供体的示意图概述。（E）Cdx2基因座处HMEJ介导的基因靶向策略的示意图概述。HAL/HAR，左/右同源臂；三角形，sgRNA靶位点；OF/OR，外部正向/反向引物；IF/IR，内部正向/反向引物。图改编自先前的报告。请点击此处查看此图的更大版本。
