Journal: PloS one

Article Title: ZO-1 knockout by TALEN-mediated gene targeting in MDCK cells: involvement of ZO-1 in the regulation of cytoskeleton and cell shape.

doi: 10.1371/journal.pone.0104994

Figure Lengend Snippet: Figure 1. Construction of TALENs and ZO-1 gene knockout in MDCK I and II cells. (A) TALEN binding sites in the ZO-1 gene. The left and right arms of TALEN targeting sites are indicated in blue and the spacer region is indicated in red. The initiation codon within the spacer region is highlighted. (B) Immunofluorescence microscopic analysis of ZO-1, ZO-2 and ZO-3 in MDCK II cells transfected with TALEN constructs for ZO-1 gene knockout. After transfection, cells were subcultured on filter inserts for 4 d before analysis. At the boundary of control and ZO-1 knockout cells, characteristic convex curves of cell–cell junctions are observed (arrows). (C) Immunofluorescence microscopic analysis of ZO-1, ZO-2 and ZO-3 in MDCK I cells transfected with TALEN constructs for ZO-1 gene knockout. Similar morphological changes of cell–cell junctions at the boundary of control and ZO-1 knockout cells were observed in MDCK I cells. Staining of ZO-3 was reduced in ZO-1 knockout cells (arrowheads). Scale bars, 10 mm. doi:10.1371/journal.pone.0104994.g001

Article Snippet: Construction of TALENs and establishment of knockout clones TALENs were constructed following the detailed instruction provided by the TALE Toolbox kit from the Zhang laboratory [27] (Addgene, #1000000019).

Techniques: TALENs, Gene Knockout, Binding Assay, Immunofluorescence, Transfection, Construct, Control, Knock-Out, Staining

Journal: PloS one

Article Title: ZO-1 knockout by TALEN-mediated gene targeting in MDCK cells: involvement of ZO-1 in the regulation of cytoskeleton and cell shape.

doi: 10.1371/journal.pone.0104994

Figure Lengend Snippet: Figure 4. Establishment of ZO-1 knockout clones in MDCK II cells. (A) Immunofluorescence microscopic analysis of ZO-1 in control (CTL) MDCK II cells and ZO-1 knockout clones (KO 1–3). ZO-1 staining was completely lost in ZO-1 knockout clones. Scale bar, 10 mm. (B) Immunoblots of ZO-1 and E-cadherin (E-cad) in control MDCK II cells and ZO-1 knockout clones. Knockout clones showed no detectable bands of ZO-1. (C) DNA sequences of TALEN targeting sites in each allele of ZO-1 knockout clones. One type of mutation was present in the alleles of ZO-1 knockout clone 1 (KO 1) and two types of mutations in the alleles of clones 2 and 3 (KO 2 and 3). Dashes indicate loss of nucleotides and green letters indicate additional nucleotides. Loss of initiating codon or frameshift were confirmed in all alleles. (D) Genomic PCR analysis of control and ZO-1 knockout clones using primers for TALENs and ZO-1 DNAs. A clone stably expressing TALEN was used as a positive control (PC). None of the PCR products for TALENs were detected in ZO-1 knockout clones. doi:10.1371/journal.pone.0104994.g004

Article Snippet: Construction of TALENs and establishment of knockout clones TALENs were constructed following the detailed instruction provided by the TALE Toolbox kit from the Zhang laboratory [27] (Addgene, #1000000019).

Techniques: Knock-Out, Clone Assay, Immunofluorescence, Control, Staining, Western Blot, Mutagenesis, TALENs, Stable Transfection, Expressing, Positive Control

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Two decades of CRISPR-Cas9 adoption and success .

Article Snippet: Bio Labs England , Q5 Hot Start High-Fidelity 2X Master Mix, NEBuilder HiFi DNA Assembly Master Mix , Q5 Site-Directed Mutagenesis Kit (with competent cells) and Q5 Site-Directed Mutagenesis Kit (Without Competent Cells) , EnGe Cas9 Nuclease , EnGen Mutation Detection Kit.

Techniques: CRISPR

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Successful application of CRISPR-Cas9 in different plant species.

Article Snippet: Bio Labs England , Q5 Hot Start High-Fidelity 2X Master Mix, NEBuilder HiFi DNA Assembly Master Mix , Q5 Site-Directed Mutagenesis Kit (with competent cells) and Q5 Site-Directed Mutagenesis Kit (Without Competent Cells) , EnGe Cas9 Nuclease , EnGen Mutation Detection Kit.

Techniques: CRISPR, Modification, Gene Knockout, Cell Surface Receptor Assay, Activity Assay, Expressing

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: How CRISPR-Cas9 perform genome editing. Cas9 induce double stranded breaks (DSBs) at particular site. The resulting DSB is then repaired by one of these two general repair pathways, e.g., by Non-homologous end joining (NHEJ) or by Homology directed repair (HDR). (A) The NHEJ repair pathway frequently results in small nucleotide insertions or deletions (InDels) at the DSB site. This may result in gene knock out or gene insertion. (B) HDR can be used to generate precise nucleotide modifications (also called gene “edits”) ranging from a single nucleotide change to large insertions.

Article Snippet: Bio Labs England , Q5 Hot Start High-Fidelity 2X Master Mix, NEBuilder HiFi DNA Assembly Master Mix , Q5 Site-Directed Mutagenesis Kit (with competent cells) and Q5 Site-Directed Mutagenesis Kit (Without Competent Cells) , EnGe Cas9 Nuclease , EnGen Mutation Detection Kit.

Techniques: CRISPR, Non-Homologous End Joining, Knock-Out

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Tabular presentation of comparative attributes of plant genome editing techniques.

Article Snippet: Bio Labs England , Q5 Hot Start High-Fidelity 2X Master Mix, NEBuilder HiFi DNA Assembly Master Mix , Q5 Site-Directed Mutagenesis Kit (with competent cells) and Q5 Site-Directed Mutagenesis Kit (Without Competent Cells) , EnGe Cas9 Nuclease , EnGen Mutation Detection Kit.

Techniques: Zinc-Fingers, TALENs, Sequencing, Clone Assay, Produced, In Vitro, Methylation, Multiplexing, CRISPR, Mutagenesis

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Technical limitations in CRISPR-Cas9 application and their effects.

Article Snippet: Bio Labs England , Q5 Hot Start High-Fidelity 2X Master Mix, NEBuilder HiFi DNA Assembly Master Mix , Q5 Site-Directed Mutagenesis Kit (with competent cells) and Q5 Site-Directed Mutagenesis Kit (Without Competent Cells) , EnGe Cas9 Nuclease , EnGen Mutation Detection Kit.

Techniques: CRISPR, Concentration Assay, Expressing, Sequencing, DNA Methylation Assay, Modification, Protein Binding

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: List of promoters and gene(s) targeted through CRISPR-Cas9 system in different plants.

Article Snippet: Bio Labs England , Q5 Hot Start High-Fidelity 2X Master Mix, NEBuilder HiFi DNA Assembly Master Mix , Q5 Site-Directed Mutagenesis Kit (with competent cells) and Q5 Site-Directed Mutagenesis Kit (Without Competent Cells) , EnGe Cas9 Nuclease , EnGen Mutation Detection Kit.

Techniques: CRISPR

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Different plasmids with their genes, vectors, and promoters used in CRISPR-Cas9 technique.

Article Snippet: Bio Labs England , Q5 Hot Start High-Fidelity 2X Master Mix, NEBuilder HiFi DNA Assembly Master Mix , Q5 Site-Directed Mutagenesis Kit (with competent cells) and Q5 Site-Directed Mutagenesis Kit (Without Competent Cells) , EnGe Cas9 Nuclease , EnGen Mutation Detection Kit.

Techniques: CRISPR, Plasmid Preparation, Expressing, Sequencing

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Diagrammatic illustration of live-cell DNA labeling by using CRISPR-Cas9 system .

Article Snippet: Bio Labs England , Q5 Hot Start High-Fidelity 2X Master Mix, NEBuilder HiFi DNA Assembly Master Mix , Q5 Site-Directed Mutagenesis Kit (with competent cells) and Q5 Site-Directed Mutagenesis Kit (Without Competent Cells) , EnGe Cas9 Nuclease , EnGen Mutation Detection Kit.

Techniques: DNA Labeling, CRISPR

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Specific commercial products and services available to the researchers to implement CRISPR technology.

Article Snippet: Bio Labs England , Q5 Hot Start High-Fidelity 2X Master Mix, NEBuilder HiFi DNA Assembly Master Mix , Q5 Site-Directed Mutagenesis Kit (with competent cells) and Q5 Site-Directed Mutagenesis Kit (Without Competent Cells) , EnGe Cas9 Nuclease , EnGen Mutation Detection Kit.

Techniques: CRISPR, Genome Wide, Clone Assay, Stable Transfection, Selection, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Negative Control, Positive Control, Construct, Knock-In, Multiplex Assay, Amplification, Sequencing

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Two decades of CRISPR-Cas9 adoption and success .

Article Snippet: Integrated DNA technologies (IDT) , Human HPRT PCR Primer Mix, Mouse HPRT PCR Primer Mix, Nuclease Free Duplex Buffer , S.p. Cas9 Expression Plasmid , S.p. Cas9 Nuclease 3NLS (100, 500 μg) , CRISPR Negative Control crRNA, CRISPR Positive Control crRNA.

Techniques: CRISPR

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Successful application of CRISPR-Cas9 in different plant species.

Article Snippet: Integrated DNA technologies (IDT) , Human HPRT PCR Primer Mix, Mouse HPRT PCR Primer Mix, Nuclease Free Duplex Buffer , S.p. Cas9 Expression Plasmid , S.p. Cas9 Nuclease 3NLS (100, 500 μg) , CRISPR Negative Control crRNA, CRISPR Positive Control crRNA.

Techniques: CRISPR, Modification, Gene Knockout, Cell Surface Receptor Assay, Activity Assay, Expressing

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: How CRISPR-Cas9 perform genome editing. Cas9 induce double stranded breaks (DSBs) at particular site. The resulting DSB is then repaired by one of these two general repair pathways, e.g., by Non-homologous end joining (NHEJ) or by Homology directed repair (HDR). (A) The NHEJ repair pathway frequently results in small nucleotide insertions or deletions (InDels) at the DSB site. This may result in gene knock out or gene insertion. (B) HDR can be used to generate precise nucleotide modifications (also called gene “edits”) ranging from a single nucleotide change to large insertions.

Article Snippet: Integrated DNA technologies (IDT) , Human HPRT PCR Primer Mix, Mouse HPRT PCR Primer Mix, Nuclease Free Duplex Buffer , S.p. Cas9 Expression Plasmid , S.p. Cas9 Nuclease 3NLS (100, 500 μg) , CRISPR Negative Control crRNA, CRISPR Positive Control crRNA.

Techniques: CRISPR, Non-Homologous End Joining, Knock-Out

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Tabular presentation of comparative attributes of plant genome editing techniques.

Article Snippet: Integrated DNA technologies (IDT) , Human HPRT PCR Primer Mix, Mouse HPRT PCR Primer Mix, Nuclease Free Duplex Buffer , S.p. Cas9 Expression Plasmid , S.p. Cas9 Nuclease 3NLS (100, 500 μg) , CRISPR Negative Control crRNA, CRISPR Positive Control crRNA.

Techniques: Zinc-Fingers, TALENs, Sequencing, Clone Assay, Produced, In Vitro, Methylation, Multiplexing, CRISPR, Mutagenesis

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Technical limitations in CRISPR-Cas9 application and their effects.

Article Snippet: Integrated DNA technologies (IDT) , Human HPRT PCR Primer Mix, Mouse HPRT PCR Primer Mix, Nuclease Free Duplex Buffer , S.p. Cas9 Expression Plasmid , S.p. Cas9 Nuclease 3NLS (100, 500 μg) , CRISPR Negative Control crRNA, CRISPR Positive Control crRNA.

Techniques: CRISPR, Concentration Assay, Expressing, Sequencing, DNA Methylation Assay, Modification, Protein Binding

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: List of promoters and gene(s) targeted through CRISPR-Cas9 system in different plants.

Article Snippet: Integrated DNA technologies (IDT) , Human HPRT PCR Primer Mix, Mouse HPRT PCR Primer Mix, Nuclease Free Duplex Buffer , S.p. Cas9 Expression Plasmid , S.p. Cas9 Nuclease 3NLS (100, 500 μg) , CRISPR Negative Control crRNA, CRISPR Positive Control crRNA.

Techniques: CRISPR

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Different plasmids with their genes, vectors, and promoters used in CRISPR-Cas9 technique.

Article Snippet: Integrated DNA technologies (IDT) , Human HPRT PCR Primer Mix, Mouse HPRT PCR Primer Mix, Nuclease Free Duplex Buffer , S.p. Cas9 Expression Plasmid , S.p. Cas9 Nuclease 3NLS (100, 500 μg) , CRISPR Negative Control crRNA, CRISPR Positive Control crRNA.

Techniques: CRISPR, Plasmid Preparation, Expressing, Sequencing

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Diagrammatic illustration of live-cell DNA labeling by using CRISPR-Cas9 system .

Article Snippet: Integrated DNA technologies (IDT) , Human HPRT PCR Primer Mix, Mouse HPRT PCR Primer Mix, Nuclease Free Duplex Buffer , S.p. Cas9 Expression Plasmid , S.p. Cas9 Nuclease 3NLS (100, 500 μg) , CRISPR Negative Control crRNA, CRISPR Positive Control crRNA.

Techniques: DNA Labeling, CRISPR

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Specific commercial products and services available to the researchers to implement CRISPR technology.

Article Snippet: Integrated DNA technologies (IDT) , Human HPRT PCR Primer Mix, Mouse HPRT PCR Primer Mix, Nuclease Free Duplex Buffer , S.p. Cas9 Expression Plasmid , S.p. Cas9 Nuclease 3NLS (100, 500 μg) , CRISPR Negative Control crRNA, CRISPR Positive Control crRNA.

Techniques: CRISPR, Genome Wide, Clone Assay, Stable Transfection, Selection, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Negative Control, Positive Control, Construct, Knock-In, Multiplex Assay, Amplification, Sequencing

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Two decades of CRISPR-Cas9 adoption and success .

Article Snippet: Integrated DNA technologies (IDT) , Human HPRT PCR Primer Mix, Mouse HPRT PCR Primer Mix, Nuclease Free Duplex Buffer , S.p. Cas9 Expression Plasmid , S.p. Cas9 Nuclease 3NLS (100, 500 μg) , CRISPR Negative Control crRNA, CRISPR Positive Control crRNA.

Techniques: CRISPR

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Successful application of CRISPR-Cas9 in different plant species.

Article Snippet: Integrated DNA technologies (IDT) , Human HPRT PCR Primer Mix, Mouse HPRT PCR Primer Mix, Nuclease Free Duplex Buffer , S.p. Cas9 Expression Plasmid , S.p. Cas9 Nuclease 3NLS (100, 500 μg) , CRISPR Negative Control crRNA, CRISPR Positive Control crRNA.

Techniques: CRISPR, Modification, Gene Knockout, Cell Surface Receptor Assay, Activity Assay, Expressing

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: How CRISPR-Cas9 perform genome editing. Cas9 induce double stranded breaks (DSBs) at particular site. The resulting DSB is then repaired by one of these two general repair pathways, e.g., by Non-homologous end joining (NHEJ) or by Homology directed repair (HDR). (A) The NHEJ repair pathway frequently results in small nucleotide insertions or deletions (InDels) at the DSB site. This may result in gene knock out or gene insertion. (B) HDR can be used to generate precise nucleotide modifications (also called gene “edits”) ranging from a single nucleotide change to large insertions.

Article Snippet: Integrated DNA technologies (IDT) , Human HPRT PCR Primer Mix, Mouse HPRT PCR Primer Mix, Nuclease Free Duplex Buffer , S.p. Cas9 Expression Plasmid , S.p. Cas9 Nuclease 3NLS (100, 500 μg) , CRISPR Negative Control crRNA, CRISPR Positive Control crRNA.

Techniques: CRISPR, Non-Homologous End Joining, Knock-Out

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Tabular presentation of comparative attributes of plant genome editing techniques.

Article Snippet: Integrated DNA technologies (IDT) , Human HPRT PCR Primer Mix, Mouse HPRT PCR Primer Mix, Nuclease Free Duplex Buffer , S.p. Cas9 Expression Plasmid , S.p. Cas9 Nuclease 3NLS (100, 500 μg) , CRISPR Negative Control crRNA, CRISPR Positive Control crRNA.

Techniques: Zinc-Fingers, TALENs, Sequencing, Clone Assay, Produced, In Vitro, Methylation, Multiplexing, CRISPR, Mutagenesis

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Technical limitations in CRISPR-Cas9 application and their effects.

Article Snippet: Integrated DNA technologies (IDT) , Human HPRT PCR Primer Mix, Mouse HPRT PCR Primer Mix, Nuclease Free Duplex Buffer , S.p. Cas9 Expression Plasmid , S.p. Cas9 Nuclease 3NLS (100, 500 μg) , CRISPR Negative Control crRNA, CRISPR Positive Control crRNA.

Techniques: CRISPR, Concentration Assay, Expressing, Sequencing, DNA Methylation Assay, Modification, Protein Binding

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: List of promoters and gene(s) targeted through CRISPR-Cas9 system in different plants.

Article Snippet: Integrated DNA technologies (IDT) , Human HPRT PCR Primer Mix, Mouse HPRT PCR Primer Mix, Nuclease Free Duplex Buffer , S.p. Cas9 Expression Plasmid , S.p. Cas9 Nuclease 3NLS (100, 500 μg) , CRISPR Negative Control crRNA, CRISPR Positive Control crRNA.

Techniques: CRISPR

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Different plasmids with their genes, vectors, and promoters used in CRISPR-Cas9 technique.

Article Snippet: Integrated DNA technologies (IDT) , Human HPRT PCR Primer Mix, Mouse HPRT PCR Primer Mix, Nuclease Free Duplex Buffer , S.p. Cas9 Expression Plasmid , S.p. Cas9 Nuclease 3NLS (100, 500 μg) , CRISPR Negative Control crRNA, CRISPR Positive Control crRNA.

Techniques: CRISPR, Plasmid Preparation, Expressing, Sequencing

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Diagrammatic illustration of live-cell DNA labeling by using CRISPR-Cas9 system .

Article Snippet: Integrated DNA technologies (IDT) , Human HPRT PCR Primer Mix, Mouse HPRT PCR Primer Mix, Nuclease Free Duplex Buffer , S.p. Cas9 Expression Plasmid , S.p. Cas9 Nuclease 3NLS (100, 500 μg) , CRISPR Negative Control crRNA, CRISPR Positive Control crRNA.

Techniques: DNA Labeling, CRISPR

Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Specific commercial products and services available to the researchers to implement CRISPR technology.

Article Snippet: Integrated DNA technologies (IDT) , Human HPRT PCR Primer Mix, Mouse HPRT PCR Primer Mix, Nuclease Free Duplex Buffer , S.p. Cas9 Expression Plasmid , S.p. Cas9 Nuclease 3NLS (100, 500 μg) , CRISPR Negative Control crRNA, CRISPR Positive Control crRNA.

Techniques: CRISPR, Genome Wide, Clone Assay, Stable Transfection, Selection, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Negative Control, Positive Control, Construct, Knock-In, Multiplex Assay, Amplification, Sequencing

Journal: Nucleic Acids Research

Article Title: Optimization of scarless human stem cell genome editing

doi: 10.1093/nar/gkt555

Figure Lengend Snippet: Functional tests of re-TALENs in human somatic and stem cells. ( a ) Schematic representation of experimental design for testing genome targeting efficiency. A genomically integrated GFP-coding sequence is disrupted by the insertion of a stop codon and a 68 bp genomic fragment derived from the AAVS1 locus (bottom). Restoration of the GFP sequence by nuclease-mediated homologous recombination with tGFP donor (top) results in GFP+ cells that can be quantitated by FACS. Re-TALENs and TALENs target identical sequences within AAVS1 fragments. ( b ) Bar graph depicting GFP+ cell percentage introduced by tGFP donor alone, TALENs with tGFP donor and re-TALENs with tGFP donor at the target locus, as measured by FACS ( N = 3, error bar = SD). Representative FACS plots are shown later in the text. ( c ) Schematic overview depicting the targeting strategy for the native AAVS1 locus. The donor plasmid, containing splicing acceptor (SA)- 2 A (self-cleaving peptides), puromycin resistant gene (PURO) and GFP were described before . The locations of PCR primers used to detect successful editing events are depicted as blue arrows. ( d ) Successfully targeted clones of PGP1 hiPSCs were selected with puromycin (0.5 µg/ml) for 2 weeks. Microscopy images of three representative GFP+ clones are shown. Cells were also stained for the pluripotency markers TRA-1-60. Scale bar: 200 µm. ( e ) PCR assays performed on these the monoclonal GFP+ hiPSC clones demonstrated successful insertions of the donor cassettes at the AAVS1 site (lanes 1–3), whereas plain hiPSCs show no evidence of successful insertion (lane C). ( f ) Sanger sequencing of the PCR amplicon from the three targeted hiPSC colonies confirmed that the expected DNA bases at the genome-insertion boundary is present.

Article Snippet: Cells were incubated in the KnockOut DMEM/F-12 medium at 37°C for 60 min using the following antibody: Anti-SSEA-4 PE (Millipore) (1: 500 diluted); Tra-1-60 (BD Pharmingen) (1:100 diluted).

Techniques: Functional Assay, TALENs, Sequencing, Derivative Assay, Homologous Recombination, Plasmid Preparation, Clone Assay, Microscopy, Staining, Amplification

Journal: Nucleic Acids Research

Article Title: Optimization of scarless human stem cell genome editing

doi: 10.1093/nar/gkt555

Figure Lengend Snippet: Using re-TALENs and ssODNs to obtain monoclonal genome-edited hiPSC without selection. ( a ) Timeline of the experiment. ( b ) Genome engineering efficiency of re-TALENs pair and ssODN (#3) assessed by the NGS platform described in b. ( c ) Sanger sequencing results of monoclonal hiPSC colonies after genome editing. Of note, the 2 bp heterogeneous genotype (CT/CT→TA/CT) was successfully introduced into the genome of PGP1-iPS-3-11, PGP1-iPS-3-13 colonies. ( d ) Immunofluorescence staining of targeted PGP1-iPS-3-11. Cells were stained for the pluripotency markers Tra-1-60 and SSEA4. ( e ) Hematoxylin and eosin staining of teratoma sections generated from monoclonal PGP1-iPS-3-11 cells.

Article Snippet: Cells were incubated in the KnockOut DMEM/F-12 medium at 37°C for 60 min using the following antibody: Anti-SSEA-4 PE (Millipore) (1: 500 diluted); Tra-1-60 (BD Pharmingen) (1:100 diluted).

Techniques: TALENs, Selection, Sequencing, Immunofluorescence, Staining, Generated