TALEN/CRISPR-Mediated eGFP Knock-In Add-On at the OCT4 Locus Does Not Impact Differentiation of Human Embryonic Stem Cells towards Endoderm
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Mendeley | Further Information

{"title"=>"TALEN/CRISPR-mediated eGFP knock-in add-on at the OCT4 locus does not impact differentiation of human embryonic stem cells towards endoderm", "type"=>"journal", "authors"=>[{"first_name"=>"Nicole A J", "last_name"=>"Krentz", "scopus_author_id"=>"55988511200"}, {"first_name"=>"Cuilan", "last_name"=>"Nian", "scopus_author_id"=>"6602624154"}, {"first_name"=>"Francis C.", "last_name"=>"Lynn", "scopus_author_id"=>"7003562203"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"scopus"=>"2-s2.0-84916219205", "sgr"=>"84916219205", "issn"=>"19326203", "doi"=>"10.1371/journal.pone.0114275", "pmid"=>"25474420", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pui"=>"600684429"}, "id"=>"af68b75b-0567-3d9c-89c5-05d635e47063", "abstract"=>"Human embryonic stem cells (hESCs) have great promise as a source of unlimited transplantable cells for regenerative medicine. However, current progress on producing the desired cell type for disease treatment has been limited due to an insufficient understanding of the developmental processes that govern their differentiation, as well as a paucity of tools to systematically study differentiation in the lab. In order to overcome these limitations, cell-type reporter hESC lines will be required. Here we outline two strategies using Transcription Activator Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-Associated protein (Cas) to create OCT4-eGFP knock-in add-on hESC lines. Thirty-one and forty-seven percent of clones were correctly modified using the TALEN and CRISPR-Cas9 systems, respectively. Further analysis of three correctly targeted clones demonstrated that the insertion of eGFP in-frame with OCT4 neither significantly impacted expression from the wild type allele nor did the fusion protein have a dramatically different biological stability. Importantly, the OCT4-eGFP fusion was easily detected using microscopy, flow cytometry and western blotting. The OCT4 reporter lines remained equally competent at producing CXCR4+ definitive endoderm that expressed a panel of endodermal genes. Moreover, the genomic modification did not impact the formation of NKX6.1+/SOX9+ pancreatic progenitor cells following directed differentiation. In conclusion, these findings demonstrate for the first time that CRISPR-Cas9 can be used to modify OCT4 and highlight the feasibility of creating cell-type specific reporter hESC lines utilizing genome-editing tools that facilitate homologous recombination.", "link"=>"http://www.mendeley.com/research/talencrisprmediated-egfp-knockin-addon-oct4-locus-not-impact-differentiation-human-embryonic-stem-ce", "reader_count"=>84, "reader_count_by_academic_status"=>{"Unspecified"=>3, "Professor > Associate Professor"=>3, "Researcher"=>18, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>23, "Student > Postgraduate"=>2, "Student > Master"=>16, "Other"=>2, "Student > Bachelor"=>12, "Lecturer"=>1, "Professor"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>3, "Professor > Associate Professor"=>3, "Researcher"=>18, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>23, "Student > Postgraduate"=>2, "Student > Master"=>16, "Other"=>2, "Student > Bachelor"=>12, "Lecturer"=>1, "Professor"=>1}, "reader_count_by_subject_area"=>{"Engineering"=>5, "Unspecified"=>4, "Biochemistry, Genetics and Molecular Biology"=>20, "Agricultural and Biological Sciences"=>45, "Medicine and Dentistry"=>4, "Pharmacology, Toxicology and Pharmaceutical Science"=>1, "Chemistry"=>3, "Immunology and Microbiology"=>1, "Chemical Engineering"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>5}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>4}, "Chemistry"=>{"Chemistry"=>3}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>45}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>20}, "Unspecified"=>{"Unspecified"=>4}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>1}, "Chemical Engineering"=>{"Chemical Engineering"=>1}}, "reader_count_by_country"=>{"Canada"=>2, "China"=>1, "United Kingdom"=>1, "Chile"=>1, "Portugal"=>1}, "group_count"=>5}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1820904"], "description"=>"<p>(A) The structure of Xanthomonas sp TALE protein. Each nucleotide-binding module is comprised of a 34 amino acid sequence, inside of which is embedded one of 4 repeat variable domains (RVD). The sequence of this di-amino acid RVD dictates the deoxynucleotide-binding cipher: NG is highly specific for deoxythymidine, HD for deoxycytidine, NI for deoxyadenosine, and NH for deoxyguanosine. (B) Schematic overview of the targeting strategy using TALENs to knock eGFP onto the OCT4 coding sequence. Red line represents the stop codon. Regions where genotyping PCR primer pairs bind are highlighted for 5′F, 5′R, 3′F and 3′R. (C) Genotyping PCR for i) 5′ arm of insertion using primers 5′F and 5′R (1180 bp) ii) 3′ arm of insertion using primers 3′F and 3′R (1000 bp) iii) endogenous allele using primers 5′F and 3′R for parent CyT49 line and three of the generated knock-in lines OCT4-2, OCT4-3 and OCT4-28. (D) Schematic overview of the CRISPR-Cas9 targeting strategy. Red line represents the stop codon. A green circle represents the Cas9 endonuclease with the tracRNA in black. The genomic protospacer adjacent motif (PAM) sequence is highlighted in red type and the guide RNA sequence is in bold type. Genotyping PCR primer pairs are the same as for TALEN targeting and are highlighted.</p>", "links"=>[], "tags"=>["differentiation", "reporter hESC lines", "Oct 4", "Clustered Regularly Interspaced", "cxcr", "talen", "OCT 4 Locus", "OCT 4 reporter lines", "human embryonic stem cells", "crispr", "nkx"], "article_id"=>1259739, "categories"=>["Biological Sciences"], "users"=>["Nicole A. J. Krentz", "Cuilan Nian", "Francis C. Lynn"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0114275.g001", "stats"=>{"downloads"=>2, "page_views"=>57, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Targeting_strategy_using_genetically_engineered_nucleases_to_generate_OCT4_eGFP_2A_Puro_hESC_lines_/1259739", "title"=>"Targeting strategy using genetically engineered nucleases to generate OCT4-eGFP-2A-Puro hESC lines.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-12-04 02:54:02"}
  • {"files"=>["https://ndownloader.figshare.com/files/1820999"], "description"=>"<p>hESCs were differentiated into definitive endoderm using a three day protocol, cells were collected and expression of OCT4 (A), CER1 (C), GSC (D), SOX7 (E), and SOX17 (F) were analyzed using Taqman qPCR. All genes were normalized to TATA Binding Protein expression (TBP). (B) hESC-derived DE cells were fixed with 4% PFA and stained for the cell surface marker CXCR4. The number of CXCR4+ DE cells was detected using BD FACSCalibur in the CyT49, OCT4-2, OCT4-3 and OCT4-28 lines. Statistical analysis was carried out using a one-way ANOVA followed by a Dunnett post-test. n≥3. *p<0.05.</p>", "links"=>[], "tags"=>["differentiation", "reporter hESC lines", "Oct 4", "Clustered Regularly Interspaced", "cxcr", "talen", "OCT 4 Locus", "OCT 4 reporter lines", "human embryonic stem cells", "crispr", "nkx"], "article_id"=>1259788, "categories"=>["Biological Sciences"], "users"=>["Nicole A. J. Krentz", "Cuilan Nian", "Francis C. Lynn"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0114275.g004", "stats"=>{"downloads"=>4, "page_views"=>37, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Differentiation_of_the_definitive_endoderm_germ_layer_is_unaffected_by_the_addition_of_eGFP_into_the_OCT4_locus_/1259788", "title"=>"Differentiation of the definitive endoderm germ layer is unaffected by the addition of eGFP into the OCT4 locus.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-12-04 02:54:02"}
  • {"files"=>["https://ndownloader.figshare.com/files/1820994"], "description"=>"<p>Cells were grown on optical dishes and then fixed with 4% PFA and permeabilized with 0.5% triton-X. Immunostaining was carried out and images were obtained on a Leica SP8 confocal microscope. Native eGFP fluorescence (green) overlapped with both SOX2 or NANOG immunostaining (red) and nuclear counterstain using TO-PRO Iodide (blue). Scale bars represent 50 µm.</p>", "links"=>[], "tags"=>["differentiation", "reporter hESC lines", "Oct 4", "Clustered Regularly Interspaced", "cxcr", "talen", "OCT 4 Locus", "OCT 4 reporter lines", "human embryonic stem cells", "crispr", "nkx"], "article_id"=>1259783, "categories"=>["Biological Sciences"], "users"=>["Nicole A. J. Krentz", "Cuilan Nian", "Francis C. Lynn"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0114275.g003", "stats"=>{"downloads"=>1, "page_views"=>41, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Stem_cell_characteristics_are_retained_in_OCT4_eGFP_2A_Puro_reporter_hESC_lines_/1259783", "title"=>"Stem cell characteristics are retained in OCT4-eGFP-2A-Puro reporter hESC lines.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-12-04 02:54:02"}
  • {"files"=>["https://ndownloader.figshare.com/files/1820963"], "description"=>"<p>Undifferentiated cells on optical dishes were fixed in 4% PFA, permeabilized using 0.5% triton-X and stained for OCT4. Images were obtained on a Leica SP8 confocal microscope and native eGFP fluorescence (green) overlapped completely with both OCT4 immunostaining (red) and nuclear counterstain using TO-PRO Iodide (blue). Scale bars represent 50 µm.</p>", "links"=>[], "tags"=>["differentiation", "reporter hESC lines", "Oct 4", "Clustered Regularly Interspaced", "cxcr", "talen", "OCT 4 Locus", "OCT 4 reporter lines", "human embryonic stem cells", "crispr", "nkx"], "article_id"=>1259764, "categories"=>["Biological Sciences"], "users"=>["Nicole A. J. Krentz", "Cuilan Nian", "Francis C. Lynn"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0114275.g002", "stats"=>{"downloads"=>2, "page_views"=>20, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Knock_in_add_on_of_eGFP_does_not_impact_native_OCT4_expression_in_targeted_hESC_lines_/1259764", "title"=>"Knock-in add on of eGFP does not impact native OCT4 expression in targeted hESC lines.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-12-04 02:54:02"}
  • {"files"=>["https://ndownloader.figshare.com/files/1821004"], "description"=>"<p>(A) The mRNA expression of OCT4 was measured during the differentiation of CyT49 cells. Day 0 represents undifferentiated hESC cells, Days 1–3 are cells becoming definitive endoderm, Days 4–6 are cells becoming posterior foregut, Day 9 are pancreatic endoderm cells and Day 12 are pancreatic progenitors and endocrine cells. (B) CyT49 cells were fixed with 4% PFA, permeabilized in 0.5% triton-X, stained using rabbit anti-OCT4 antibodies followed by FITC-conjugated donkey anti-rabbit antibodies. The number of FITC+ cells was measured using a BD FACSCalibur during several days of the differentiation protocol. The number of GFP<sup>+</sup> cells was measured using native eGFP fluorescence and a BD FACSCalibur in OCT4-2 (C), OCT4-3 (D) and OCT4-28 (E) fixed cells. n≥3.</p>", "links"=>[], "tags"=>["differentiation", "reporter hESC lines", "Oct 4", "Clustered Regularly Interspaced", "cxcr", "talen", "OCT 4 Locus", "OCT 4 reporter lines", "human embryonic stem cells", "crispr", "nkx"], "article_id"=>1259793, "categories"=>["Biological Sciences"], "users"=>["Nicole A. J. Krentz", "Cuilan Nian", "Francis C. Lynn"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0114275.g005", "stats"=>{"downloads"=>0, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_GFP_expression_decreases_upon_differentiation_towards_pancreatic_progenitor_cells_/1259793", "title"=>"GFP expression decreases upon differentiation towards pancreatic progenitor cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-12-04 02:54:02"}
  • {"files"=>["https://ndownloader.figshare.com/files/1821043"], "description"=>"<p>Immunostaining for NKX6-1 (green) and SOX9 (red) was carried out on day 12 in parent CyT49 hESCs and knock-in OCT4-2, OCT4-3 and OCT4-28 hESC lines that were grown and differentiated in optical dishes. Images were obtained on a Leica SP8 confocal microscope using TO-PRO Iodide (blue) as nuclear counterstain. Scale bars represent 25 µm.</p>", "links"=>[], "tags"=>["differentiation", "reporter hESC lines", "Oct 4", "Clustered Regularly Interspaced", "cxcr", "talen", "OCT 4 Locus", "OCT 4 reporter lines", "human embryonic stem cells", "crispr", "nkx"], "article_id"=>1259822, "categories"=>["Biological Sciences"], "users"=>["Nicole A. J. Krentz", "Cuilan Nian", "Francis C. Lynn"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0114275.g006", "stats"=>{"downloads"=>0, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Addition_of_eGFP_does_not_affect_the_efficiency_of_pancreatic_progenitor_formation_during_in_vitro_differentiation_protocol_/1259822", "title"=>"Addition of eGFP does not affect the efficiency of pancreatic progenitor formation during <i>in vitro</i> differentiation protocol.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-12-04 02:54:02"}
  • {"files"=>["https://ndownloader.figshare.com/files/1821076", "https://ndownloader.figshare.com/files/1821077", "https://ndownloader.figshare.com/files/1821078", "https://ndownloader.figshare.com/files/1821079", "https://ndownloader.figshare.com/files/1821081"], "description"=>"<div><p>Human embryonic stem cells (hESCs) have great promise as a source of unlimited transplantable cells for regenerative medicine. However, current progress on producing the desired cell type for disease treatment has been limited due to an insufficient understanding of the developmental processes that govern their differentiation, as well as a paucity of tools to systematically study differentiation in the lab. In order to overcome these limitations, cell-type reporter hESC lines will be required. Here we outline two strategies using Transcription Activator Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-Associated protein (Cas) to create OCT4-eGFP knock-in add-on hESC lines. Thirty-one and forty-seven percent of clones were correctly modified using the TALEN and CRISPR-Cas9 systems, respectively. Further analysis of three correctly targeted clones demonstrated that the insertion of eGFP in-frame with OCT4 neither significantly impacted expression from the wild type allele nor did the fusion protein have a dramatically different biological stability. Importantly, the OCT4-eGFP fusion was easily detected using microscopy, flow cytometry and western blotting. The OCT4 reporter lines remained equally competent at producing CXCR4+ definitive endoderm that expressed a panel of endodermal genes. Moreover, the genomic modification did not impact the formation of NKX6.1+/SOX9+ pancreatic progenitor cells following directed differentiation. In conclusion, these findings demonstrate for the first time that CRISPR-Cas9 can be used to modify <i>OCT4</i> and highlight the feasibility of creating cell-type specific reporter hESC lines utilizing genome-editing tools that facilitate homologous recombination.</p></div>", "links"=>[], "tags"=>["differentiation", "reporter hESC lines", "Oct 4", "Clustered Regularly Interspaced", "cxcr", "talen", "OCT 4 Locus", "OCT 4 reporter lines", "human embryonic stem cells", "crispr", "nkx"], "article_id"=>1259844, "categories"=>["Biological Sciences"], "users"=>["Nicole A. J. Krentz", "Cuilan Nian", "Francis C. Lynn"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0114275.s001", "https://dx.doi.org/10.1371/journal.pone.0114275.s002", "https://dx.doi.org/10.1371/journal.pone.0114275.s003", "https://dx.doi.org/10.1371/journal.pone.0114275.s004", "https://dx.doi.org/10.1371/journal.pone.0114275.s005"], "stats"=>{"downloads"=>14, "page_views"=>48, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/TALEN_CRISPR_Mediated_eGFP_Knock_In_Add_On_at_the_OCT4_Locus_Does_Not_Impact_Differentiation_of_Human_Embryonic_Stem_Cells_towards_Endoderm/1259844", "title"=>"TALEN/CRISPR-Mediated eGFP Knock-In Add-On at the <i>OCT4</i> Locus Does Not Impact Differentiation of Human Embryonic Stem Cells towards Endoderm", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2014-12-04 02:54:02"}
  • {"files"=>["https://ndownloader.figshare.com/files/1821047"], "description"=>"<p>Targeting efficiency of TALEN/CRISPR-mediated OCT4-eGFP-2A-Puro CyT49 hESC lines.</p>", "links"=>[], "tags"=>["differentiation", "reporter hESC lines", "Oct 4", "Clustered Regularly Interspaced", "cxcr", "talen", "OCT 4 Locus", "OCT 4 reporter lines", "human embryonic stem cells", "crispr", "nkx"], "article_id"=>1259826, "categories"=>["Biological Sciences"], "users"=>["Nicole A. J. Krentz", "Cuilan Nian", "Francis C. Lynn"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0114275.t001", "stats"=>{"downloads"=>7, "page_views"=>38, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Targeting_efficiency_of_TALEN_CRISPR_mediated_OCT4_eGFP_2A_Puro_CyT49_hESC_lines_/1259826", "title"=>"Targeting efficiency of TALEN/CRISPR-mediated OCT4-eGFP-2A-Puro CyT49 hESC lines.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-12-04 02:54:02"}

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Relative Metric

{"start_date"=>"2014-01-01T00:00:00Z", "end_date"=>"2014-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Biology and life sciences", "average_usage"=>[291]}, {"subject_area"=>"/Biology and life sciences/Biochemistry", "average_usage"=>[282]}, {"subject_area"=>"/Biology and life sciences/Biotechnology", "average_usage"=>[310, 499]}, {"subject_area"=>"/Engineering and technology", "average_usage"=>[282]}, {"subject_area"=>"/Engineering and technology/Synthetic biology", "average_usage"=>[386, 689]}]}
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