cas9 egfp expressing vector  (Addgene inc)

Journal: Cell Research

Article Title: Homology-mediated end joining-based targeted integration using CRISPR/Cas9

doi: 10.1038/cr.2017.76

Figure Lengend Snippet: In vitro genome editing via HMEJ-mediated targeted integration (A) Schematic overview of four gene targeting strategies at the Actb locus. HAL/HAR, left/right homology arm; triangles, sgRNA target sites; OF/OR, outer forward/reverse primer; IF/IR, inner forward/reverse primer. (B) Experimental scheme for targeted Actb -2A-mCherry knock-in in mouse ES cells. Cells were transfected with donors/sgRNA/GFP or donors/sgRNA/mCherry and Cas9, and transfected cells were sorted based on GFP or mCherry signals 2 days after transfections. Knock-in efficiencies were evaluated by FACS based on the ratio of GFP + or mCherry + cells among total transfected cells 4 days after the first sorting. (C) Relative knock-in efficiency of HR-, NHEJ-, MMEJ- and HMEJ-based strategies in mouse ES cells at various loci measured by the percentage of mCherry + (or GFP + ) cells among total transfected cells. Note that the NHEJ-based method was not performed at Sox2 , Nanog and Rosa26 loci. (D) Schematic overview of insertion fragments at different loci. (E , F) Relative knock-in efficiency of HR-, NHEJ-, MMEJ- and HMEJ-based strategies in N2a cells (E) and HEK293T cells (F) at various loci measured by the percentage of mCherry + (or GFP + ) cells among total transfected cells. Note that the NHEJ-based method was not performed at the Rosa26 locus. (G) Relative knock-in efficiency in primary astrocytes and neurons measured by the percentage of mCherry + cells among GFP + cells. The results in panels C , E , F and G were presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, unpaired Student's t -test.

Article Snippet: To generate a single Cas9-sgRNA-EGFP expressing vector, a modified pX330 (Addgene catalog no. 42230) expression vector expressing Cas9-CMV-EGFP and sgRNA was linearized with Bbs I digestion, and gel purified.

Techniques: In Vitro, Knock-In, Transfection

Journal: Cell Research

Article Title: Homology-mediated end joining-based targeted integration using CRISPR/Cas9

doi: 10.1038/cr.2017.76

Figure Lengend Snippet: HMEJ-mediated targeted integration in mouse embryos. (A) Experimental design. Cas9 mRNA, sgRNA and donor vector were injected into mouse zygotes and the injected zygotes were cultured to the blastocyst stage to observe fluorescence and for genotyping analysis. (B) Representative immunofluorescence images of gene-edited mouse embryos at the blastocyst stage. Cas9 mRNA, sgRNA and each donor vector (HR, MMEJ or HMEJ) were injected into mouse zygotes and the injected zygotes were cultured to the blastocyst stage for fluorescence observation. The control, HMEJ donor without Cas9. Insets, higher magnification images. Scale bar, 50 μm. (C) Knock-in efficiencies indicated by percentage of mCherry + blastocysts. Number above each bar, total blastocysts counted. (D) Efficiencies of mice with p2A-mCherry precise integration at Sox2 and Dbh loci. Number above each bar, total mice counted. C and D , * P < 0.05, ** P < 0.01, *** P < 0.001, χ 2 -test. (E) Representative immunofluorescence images of brain in 6-week-old mice with Dbh -p2A-mCherry knock-in by HMEJ-based method. Scale bar, 50 μm. Arrowheads, TE; Asterisk, ICM; TH, tyrosine hydroxylase.

Article Snippet: To generate a single Cas9-sgRNA-EGFP expressing vector, a modified pX330 (Addgene catalog no. 42230) expression vector expressing Cas9-CMV-EGFP and sgRNA was linearized with Bbs I digestion, and gel purified.

Techniques: Plasmid Preparation, Injection, Cell Culture, Fluorescence, Immunofluorescence, Knock-In

Journal: Cell Research

Article Title: Homology-mediated end joining-based targeted integration using CRISPR/Cas9

doi: 10.1038/cr.2017.76

Figure Lengend Snippet: HMEJ-mediated targeted integration in monkey embryos. (A) Schematic overview of HMEJ-mediated gene targeting strategy at the Actb locus in monkey embryos. Intracytoplasmic sperm injection (ICSI) was performed on monkey oocyte and the Cas9 mix was injected 6 h later. The injected embryos were cultured for 7 days into blastocysts. (B) Representative immunofluorescence images of gene-edited monkey embryos at the blastocyst stage. Square, blastocysts shown at a higher resolution on the right panel. Scale bar, 100 μm. Numbered blastocysts were genotyped and labeled with * below. (C) Knock-in efficiencies with different concentrations of donor plasmids (100, 50 ng/μl) indicated by percentage of mCherry + blastocysts. Number above the bar, total blastocysts counted. (D) Genotyping analysis of the injected embryos. PCR products amplified from 5′ and 3′ junction sites of DNA samples of individual monkey embryos on day 7 were sequenced and shown in D . PC, positive control from COS-7 cells with Actb -p2A-mCherry knock-in. * , mcherry + blastocyst shown in B . (E) Genotype of integration junctions in total injected embryos. Number above the bar, total embryos analysis. (F) Sequencing analysis of integration junctions of the injected embryos. PCR products amplified from the 5′ and 3′ junction sites of DNA samples extracted from individual embryos were sequenced. Number, total sample size. CCT to GGT, replace PAM sequence CCT of sgRNA to GGT to avoid recutting. Dashed lines mark the region omitted for clarity. Upper, homology arm; purple, intron 4; red, mCherry; green, PAM sequence. Dashed lines mark the region omitted for clarity.

Article Snippet: To generate a single Cas9-sgRNA-EGFP expressing vector, a modified pX330 (Addgene catalog no. 42230) expression vector expressing Cas9-CMV-EGFP and sgRNA was linearized with Bbs I digestion, and gel purified.

Techniques: Injection, Cell Culture, Immunofluorescence, Labeling, Knock-In, Amplification, Positive Control, Sequencing