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  • 99
    Thermo Fisher gene expression cassettes
    Gene Expression Cassettes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene expression cassettes/product/Thermo Fisher
    Average 99 stars, based on 35 article reviews
    Price from $9.99 to $1999.99
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    99
    Millipore short hairpin rna expression cassette
    <t>M6P/IGF2R</t> expression increases during monocyte differentiation to macrophages . ( A ) Cell‐surface expression of M6P/IGF2R on isolated PBMC and MACS‐enriched monocytes from healthy donors was evaluated with mAb MEM‐238‐AF647 and flow cytometry. In parallel, MOPC‐21‐AF647 was used as an isotype control mAb, displayed by the cut‐off gates. The same analysis was performed with macrophages differentiated from MACS‐sorted monocytes during a 7‐day culture with recombinant human M‐CSF (50 ng/ml) followed by 2 days resting in macrophage serum free medium. ( B ) Primary human monocytes and monocyte‐derived macrophages from (A) were lysed and <t>RNA</t> was extracted. cDNA was synthesized from total RNA and gene expression was measured by real‐time PCR as described in the Material and Methods section with TaqMan primer sets for human M6P/IGF2R and YWHAZ as endogenous control. The M6P/IGF2R mean expression values relative to that of monocytes ± SD from 3 donors is shown
    Short Hairpin Rna Expression Cassette, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/short hairpin rna expression cassette/product/Millipore
    Average 99 stars, based on 10 article reviews
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    88
    Addgene inc cas9 expression cassette
    Typical experimental workflow for GO-CRISPR screening and analysis using TRACS. (A) iOvCa147 High-grade serous ovarian cancer (HGSOC) cells were transduced with lentivirus expressing <t>Cas9.</t> High efficiency Cas9-positive cells (top row) and Cas9-negative cells (bottom row) were transduced with the GeCKO v2 pooled sgRNA library (L 0 ). After antibiotic selection, both Cas9 positive and negative cells were split into triplicates (x3) and maintained in initial culture conditions (T 0 ) before being transferred to suspension culture conditions in ULA plasticware (selective pressure, P s ) to induce spheroid formation and select for cell survival. Viable spheroid cells were then transferred to standard plasticware to facilitate reattachment in the final culture condition (T f ). The initial pooled sgRNA library (L 0 ) and Cas9-positive and Cas9-negative cells were collected at T 0 and T f for sgRNA quantitation by NGS. TRACS was used to calculate Library, Initial and Final Enrichment Scores (ES) using read quantities from L 0 and Cas9-positive and Cas9-negative samples. (B) 3D plot output from TRACS illustrating the Library ES, Initial ES and Final ES for each gene. Genes highlighted in dark blue have low Library ES (determined by calculating the first quartile value across all Library ES;
    Cas9 Expression Cassette, supplied by Addgene inc, used in various techniques. Bioz Stars score: 88/100, based on 102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cas9 expression cassette/product/Addgene inc
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    91
    Addgene inc sgrna expression cassette
    Enhancement of knock-in efficiency with the LoAD system. a Schematic of conventional overexpression and the LoAD system. Effector proteins such as CtIP can accumulate at the DSB site via the interaction between <t>MS2</t> RNA loop and MS2 coat protein, resulting in programmable genome editing. <t>sgRNA</t> MS2 , sgRNA with two MS2 RNA loops. b Schematic of PITCh-mediated knock-in of fluorescent protein gene, harnessing MMEJ for the predefined, seamless gene cassette integration. This schematic only shows gene targeting in the CANX locus, but other gene loci such as ATB5B , PARP1 , FBL , and RPL11 were targeted in a similar way. c Knock-in frequency at the CANX locus measured by FACS analysis. The recruitment of MS2-CtIP with the LoAD system resulted in about 50% gene knock-in without any selection. Data are expressed as means ± s.e.m. ( n = 3). ** P
    Sgrna Expression Cassette, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Promega firefly luciferase expression cassette
    Enhancement of knock-in efficiency with the LoAD system. a Schematic of conventional overexpression and the LoAD system. Effector proteins such as CtIP can accumulate at the DSB site via the interaction between <t>MS2</t> RNA loop and MS2 coat protein, resulting in programmable genome editing. <t>sgRNA</t> MS2 , sgRNA with two MS2 RNA loops. b Schematic of PITCh-mediated knock-in of fluorescent protein gene, harnessing MMEJ for the predefined, seamless gene cassette integration. This schematic only shows gene targeting in the CANX locus, but other gene loci such as ATB5B , PARP1 , FBL , and RPL11 were targeted in a similar way. c Knock-in frequency at the CANX locus measured by FACS analysis. The recruitment of MS2-CtIP with the LoAD system resulted in about 50% gene knock-in without any selection. Data are expressed as means ± s.e.m. ( n = 3). ** P
    Firefly Luciferase Expression Cassette, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 98 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/firefly luciferase expression cassette/product/Promega
    Average 88 stars, based on 98 article reviews
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    firefly luciferase expression cassette - by Bioz Stars, 2020-09
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    93
    GenScript expression cassettes
    Enhancement of knock-in efficiency with the LoAD system. a Schematic of conventional overexpression and the LoAD system. Effector proteins such as CtIP can accumulate at the DSB site via the interaction between <t>MS2</t> RNA loop and MS2 coat protein, resulting in programmable genome editing. <t>sgRNA</t> MS2 , sgRNA with two MS2 RNA loops. b Schematic of PITCh-mediated knock-in of fluorescent protein gene, harnessing MMEJ for the predefined, seamless gene cassette integration. This schematic only shows gene targeting in the CANX locus, but other gene loci such as ATB5B , PARP1 , FBL , and RPL11 were targeted in a similar way. c Knock-in frequency at the CANX locus measured by FACS analysis. The recruitment of MS2-CtIP with the LoAD system resulted in about 50% gene knock-in without any selection. Data are expressed as means ± s.e.m. ( n = 3). ** P
    Expression Cassettes, supplied by GenScript, used in various techniques. Bioz Stars score: 93/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 54 article reviews
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    88
    Thermo Fisher u6 shrna expression cassette
    Enhancement of knock-in efficiency with the LoAD system. a Schematic of conventional overexpression and the LoAD system. Effector proteins such as CtIP can accumulate at the DSB site via the interaction between <t>MS2</t> RNA loop and MS2 coat protein, resulting in programmable genome editing. <t>sgRNA</t> MS2 , sgRNA with two MS2 RNA loops. b Schematic of PITCh-mediated knock-in of fluorescent protein gene, harnessing MMEJ for the predefined, seamless gene cassette integration. This schematic only shows gene targeting in the CANX locus, but other gene loci such as ATB5B , PARP1 , FBL , and RPL11 were targeted in a similar way. c Knock-in frequency at the CANX locus measured by FACS analysis. The recruitment of MS2-CtIP with the LoAD system resulted in about 50% gene knock-in without any selection. Data are expressed as means ± s.e.m. ( n = 3). ** P
    U6 Shrna Expression Cassette, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/u6 shrna expression cassette/product/Thermo Fisher
    Average 88 stars, based on 32 article reviews
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    u6 shrna expression cassette - by Bioz Stars, 2020-09
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    89
    Genechem gfp expression cassette
    No effects of CatD upregulation on HK2 proliferation under culture with normal glucose. (A) HK2 cells were infected with <t>lentiviral</t> vectors containing empty or CatD construct for 12 h, then cultured for additional 48 h. Transfection efficiency was analyzed by measurement of <t>GFP+</t> positive cells under a fluorescence microscopy, and representative images are provided. (B) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h, western blot was performed using the indicated antibodies. (C) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h. Viable cells were counted using trypan blue exclusion experiment daily. (D) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h. Cell proliferation was analyzed using RTCA in a real-time pattern. (E) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h. DNA synthesis was analyzed using the EdU incorporation assay, and representative images are provided. (F) Total and EdU-positive cells in (E) were counted, and the rate of EdU incorporation was calculated. (G) HK2 cells were infected with lentiviral vectors containing empty or CatD construct, and cell cycle progression was analyzed using flowcytometry. N.S., not significant.
    Gfp Expression Cassette, supplied by Genechem, used in various techniques. Bioz Stars score: 89/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gfp expression cassette/product/Genechem
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    99
    Millipore shrna expression cassettes
    No effects of CatD upregulation on HK2 proliferation under culture with normal glucose. (A) HK2 cells were infected with <t>lentiviral</t> vectors containing empty or CatD construct for 12 h, then cultured for additional 48 h. Transfection efficiency was analyzed by measurement of <t>GFP+</t> positive cells under a fluorescence microscopy, and representative images are provided. (B) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h, western blot was performed using the indicated antibodies. (C) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h. Viable cells were counted using trypan blue exclusion experiment daily. (D) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h. Cell proliferation was analyzed using RTCA in a real-time pattern. (E) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h. DNA synthesis was analyzed using the EdU incorporation assay, and representative images are provided. (F) Total and EdU-positive cells in (E) were counted, and the rate of EdU incorporation was calculated. (G) HK2 cells were infected with lentiviral vectors containing empty or CatD construct, and cell cycle progression was analyzed using flowcytometry. N.S., not significant.
    Shrna Expression Cassettes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Genechem 4 1bb cd3ζ expression cassette
    No effects of CatD upregulation on HK2 proliferation under culture with normal glucose. (A) HK2 cells were infected with <t>lentiviral</t> vectors containing empty or CatD construct for 12 h, then cultured for additional 48 h. Transfection efficiency was analyzed by measurement of <t>GFP+</t> positive cells under a fluorescence microscopy, and representative images are provided. (B) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h, western blot was performed using the indicated antibodies. (C) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h. Viable cells were counted using trypan blue exclusion experiment daily. (D) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h. Cell proliferation was analyzed using RTCA in a real-time pattern. (E) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h. DNA synthesis was analyzed using the EdU incorporation assay, and representative images are provided. (F) Total and EdU-positive cells in (E) were counted, and the rate of EdU incorporation was calculated. (G) HK2 cells were infected with lentiviral vectors containing empty or CatD construct, and cell cycle progression was analyzed using flowcytometry. N.S., not significant.
    4 1bb Cd3ζ Expression Cassette, supplied by Genechem, used in various techniques. Bioz Stars score: 92/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/4 1bb cd3ζ expression cassette/product/Genechem
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    99
    Thermo Fisher silencer express sirna expression cassette kit
    Effect of shRNAs 1 and 2 on MCMV replication at m .o.i. = 0.2. a) An <t>SEC</t> expressing <t>shRNA-2</t> was transfected into M2-10B4 cells 24 hrs prior to infection with MCMV at an m.o.i. of 0.2. b) Western blot of IE-3 protein levels in shRNA-2 treated and control shRNA treated samples Effect of MCMV specific shRNA at low m.o.i.'s in vitro . c) Virus titers at day 5 p.i. in M2-10B4 cultures infected with MCMV at the indicated m.o.i.'s. Dark bars: 50 nM IE-3 specific shRNA-1, white bars: control shRNA. d) Western blot showing the time course of IE-3 protein expression in MCMV-infected M2-10B4 cells (m.o.i. = 0.2) following transfection of the IE-3 shRNA-1 expressing plasmid. e) Viral titers at day 5 p.i. in M2-10B4 cultures infected with MCMV at various m.o.i.'s and treated with either shRNA-1a or shRNA-2a.
    Silencer Express Sirna Expression Cassette Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Evrogen tagbfp expression cassette
    Construction and analysis of pHSVF-CREin. a The infectious clone pHSVF-CREin was made by insertion of the CREin expression cassette into the pHSVF-BFP infectious clone in a process paralleling that described in Fig. 1 . The CREin expression cassette was PCR amplified from the pEP-CREin-in template and recombined into pHSVF-BFP BAC vector backbone by lambda RED recombination, resulting in the replacement of the <t>TagBFP</t> coding sequence with that of CREin::kanamycin. In the second recombination step, the kanamycin resistance gene (aphA1) was removed based on partially duplicated sequences in the flanking CREin coding sequence ( red boxes ), which simultaneously established the contiguous CREin coding sequence and resulted in pHSVF-CREin. Excision of the BAC from pHSVF-CREin was achieved by autonomous expression of Cre recombinase following introduction of the DNA into either Vero or HEK293T cells, resulting in a passage 1 (P1) harvest. b MluI ( left ) or PvuI ( right ) restriction analysis of <t>HSV-1</t> DNA harvested from purified nucleocapsids. Yellow arrow heads indicate restriction fragments that mark the presence or absence of the BAC vector sequences. Size standards are indicated in kb. c Amplification of viral and plasmid DNA using primers designed to detect removal of BAC DNA from the viral genome. The positions of the primer pairs are indicated in panel A
    Tagbfp Expression Cassette, supplied by Evrogen, used in various techniques. Bioz Stars score: 90/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher acriia4 expression cassettes
    In vivo dCas9 association assay for anti-CRISPR proteins. (a) A yeast strain was constructed harbouring an inducible dCas9 (D10A H840A) fused to both mCherry and LactC2 at its C-terminus (GFY-3104) and transformed with (i) a sgRNA[u1] plasmid (pGF-V1220) and (ii) GFP-tagged AcrIIA2/A4-containing plasmids (pGF-IVL1384 to pGF-IVL1387). Strains were cultured overnight in raffinose/sucrose medium lacking uracil and lacking leucine, back-diluted into synthetic medium containing galactose also lacking uracil and leucine, and grown for 4.5 h at 30 °C. Cells were harvested, washed with water and imaged by fluorescence microscopy. Representative images are displayed. Scale bar, 3 µm. (b) A measure of the ratio between the maximum pixel intensity located on the PM was compared to a sampling of the average cytosolic pixel intensity for the GFP signal ( n =15–30 cells per genotype). Ten random individual measurements were taken for both the PM and cytosolic levels per cell. Error, sd . Three additional <t>AcrIIA4</t> constructs were tested (asterisks) containing sets of two alanine substitutions (pGF-IVL1431 to pGF-IVL1433). Bottom , select strains were compared using an unpaired t -test. Red text, P -values greater than 0.05.
    Acriia4 Expression Cassettes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GenScript synthetic aav2 expression cassette
    AAV Vector Delivery of Tissue Optimized Transgene Cassettes (A) Schematics of <t>AAV2/8</t> transgene designs. Straight lines represent DNA sequence of no known function. (B) AAV2/8-HLP-An53-LCO and AAV2/8-HLP-An53-HCO were delivered intravenously to hemophilia A mice at a dose of 1 × 10 11 vg/kg (n = 4 per group). (C) AAV2/8 HCB-HSQ-LCO, AAV2/8-HCB-ET3-LCO, and AAV2/8-HLP-V3co were delivered intravenously to hemophilia A mice at a dose of 1 × 10 11 vg/kg (n = 3–4 per group). (D) A long-term, dose-finding experiment was performed with varying does of AAV2/8-HCB-MVM-ET3-LCO (n = 3 for all other doses). Data are presented as the mean ± sample SD.
    Synthetic Aav2 Expression Cassette, supplied by GenScript, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    synthetic aav2 expression cassette - by Bioz Stars, 2020-09
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    99
    Thermo Fisher synthetic mirna hairpin constructs expression cassettes
    AAV Vector Delivery of Tissue Optimized Transgene Cassettes (A) Schematics of <t>AAV2/8</t> transgene designs. Straight lines represent DNA sequence of no known function. (B) AAV2/8-HLP-An53-LCO and AAV2/8-HLP-An53-HCO were delivered intravenously to hemophilia A mice at a dose of 1 × 10 11 vg/kg (n = 4 per group). (C) AAV2/8 HCB-HSQ-LCO, AAV2/8-HCB-ET3-LCO, and AAV2/8-HLP-V3co were delivered intravenously to hemophilia A mice at a dose of 1 × 10 11 vg/kg (n = 3–4 per group). (D) A long-term, dose-finding experiment was performed with varying does of AAV2/8-HCB-MVM-ET3-LCO (n = 3 for all other doses). Data are presented as the mean ± sample SD.
    Synthetic Mirna Hairpin Constructs Expression Cassettes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher expression cassettes
    AAV Vector Delivery of Tissue Optimized Transgene Cassettes (A) Schematics of <t>AAV2/8</t> transgene designs. Straight lines represent DNA sequence of no known function. (B) AAV2/8-HLP-An53-LCO and AAV2/8-HLP-An53-HCO were delivered intravenously to hemophilia A mice at a dose of 1 × 10 11 vg/kg (n = 4 per group). (C) AAV2/8 HCB-HSQ-LCO, AAV2/8-HCB-ET3-LCO, and AAV2/8-HLP-V3co were delivered intravenously to hemophilia A mice at a dose of 1 × 10 11 vg/kg (n = 3–4 per group). (D) A long-term, dose-finding experiment was performed with varying does of AAV2/8-HCB-MVM-ET3-LCO (n = 3 for all other doses). Data are presented as the mean ± sample SD.
    Expression Cassettes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1013 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 91 stars, based on 1013 article reviews
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    92
    Thermo Fisher be3 expression cassette
    BE-FLARE facilitates reciprocal enrichment of co-edited cells. a Enrichment for BFP editing by selection for EGFR T790 M co-editing. PC9 cells stably expressing the BE-FLARE reporter were co-transfected with a construct expressing <t>BE3</t> and gRNAs targeting EGFR T790 and BFP. Cells were treated with 100 nM gefitinib 72 h after transfection to select for EGFR T790 M mutants. GFP-positive cells were quantified by flow cytometry 4 days after addition of gefitinib. b Quantification of three independent experiments from ( a ), with each data point shown and mean represented as a bar. Unpaired Student’s t test; *** P
    Be3 Expression Cassette, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GenScript cellulase expression cassette
    BE-FLARE facilitates reciprocal enrichment of co-edited cells. a Enrichment for BFP editing by selection for EGFR T790 M co-editing. PC9 cells stably expressing the BE-FLARE reporter were co-transfected with a construct expressing <t>BE3</t> and gRNAs targeting EGFR T790 and BFP. Cells were treated with 100 nM gefitinib 72 h after transfection to select for EGFR T790 M mutants. GFP-positive cells were quantified by flow cytometry 4 days after addition of gefitinib. b Quantification of three independent experiments from ( a ), with each data point shown and mean represented as a bar. Unpaired Student’s t test; *** P
    Cellulase Expression Cassette, supplied by GenScript, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Addgene inc luciferase expression cassette
    BE-FLARE facilitates reciprocal enrichment of co-edited cells. a Enrichment for BFP editing by selection for EGFR T790 M co-editing. PC9 cells stably expressing the BE-FLARE reporter were co-transfected with a construct expressing <t>BE3</t> and gRNAs targeting EGFR T790 and BFP. Cells were treated with 100 nM gefitinib 72 h after transfection to select for EGFR T790 M mutants. GFP-positive cells were quantified by flow cytometry 4 days after addition of gefitinib. b Quantification of three independent experiments from ( a ), with each data point shown and mean represented as a bar. Unpaired Student’s t test; *** P
    Luciferase Expression Cassette, supplied by Addgene inc, used in various techniques. Bioz Stars score: 88/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/luciferase expression cassette/product/Addgene inc
    Average 88 stars, based on 9 article reviews
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    92
    Icosagen plasmid expression cassettes
    BE-FLARE facilitates reciprocal enrichment of co-edited cells. a Enrichment for BFP editing by selection for EGFR T790 M co-editing. PC9 cells stably expressing the BE-FLARE reporter were co-transfected with a construct expressing <t>BE3</t> and gRNAs targeting EGFR T790 and BFP. Cells were treated with 100 nM gefitinib 72 h after transfection to select for EGFR T790 M mutants. GFP-positive cells were quantified by flow cytometry 4 days after addition of gefitinib. b Quantification of three independent experiments from ( a ), with each data point shown and mean represented as a bar. Unpaired Student’s t test; *** P
    Plasmid Expression Cassettes, supplied by Icosagen, used in various techniques. Bioz Stars score: 92/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 30 article reviews
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    99
    TaKaRa puromycin resistance expression cassette
    BE-FLARE facilitates reciprocal enrichment of co-edited cells. a Enrichment for BFP editing by selection for EGFR T790 M co-editing. PC9 cells stably expressing the BE-FLARE reporter were co-transfected with a construct expressing <t>BE3</t> and gRNAs targeting EGFR T790 and BFP. Cells were treated with 100 nM gefitinib 72 h after transfection to select for EGFR T790 M mutants. GFP-positive cells were quantified by flow cytometry 4 days after addition of gefitinib. b Quantification of three independent experiments from ( a ), with each data point shown and mean represented as a bar. Unpaired Student’s t test; *** P
    Puromycin Resistance Expression Cassette, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 17 article reviews
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    88
    Addgene inc grna expression cassette
    Activation of the mouse Oct4 and Nanog loci by TALE-As and dCas9-As/gRNAs. ( A ) Schematic diagram of the dCas9-As evaluated in this study. In all cases, blue florescent protein (BFP) was used to track the expression of dCas9-As. <t>gRNA</t> expression is controlled by <t>U6</t> promoter, and EF1a-mCherry is used to detect the integration of the vector into the genome. A blasticidin resistance and the reverse tetracycline transactivator (rtTA) cassettes were also linked to the mCherry cassette by T2A peptides. PB-5TR and PB-3TR are the two terminal repeat sequences of the piggyBac (PB) transposon. ( B ) Schematic diagram of the TALE and dCas9/gRNA targeting sites at the mouse Oct4 and Nanog enhancers. Red arrows indicate the gRNA targeting sites and the blue arrows mark the TALE sites. ( C ) Activation of the Oct4 distal enhancer luciferase reporter by the TALE-As and dCas9-As/gRNAs. ( D ) qRT-PCR analysis of the Oct4 mRNA levels in MEFs expressing the TALE-As or dCas9-As/gRNAs. ( E ) Activation of the Nanog enhancer luciferase reporter by the TALE-As and dCas9-As/gRNAs. ( F ) qRT-PCR analysis of the Nanog mRNA levels in EpiSCs expressing the TALE-As or dCas9-As/gRNAs. All gene expression values were normalized to Gapdh . Results were representative of three independent experiments and were presented as ±SD, n = 3. * P
    Grna Expression Cassette, supplied by Addgene inc, used in various techniques. Bioz Stars score: 88/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Addgene inc shrna expression cassette
    Activation of the mouse Oct4 and Nanog loci by TALE-As and dCas9-As/gRNAs. ( A ) Schematic diagram of the dCas9-As evaluated in this study. In all cases, blue florescent protein (BFP) was used to track the expression of dCas9-As. <t>gRNA</t> expression is controlled by <t>U6</t> promoter, and EF1a-mCherry is used to detect the integration of the vector into the genome. A blasticidin resistance and the reverse tetracycline transactivator (rtTA) cassettes were also linked to the mCherry cassette by T2A peptides. PB-5TR and PB-3TR are the two terminal repeat sequences of the piggyBac (PB) transposon. ( B ) Schematic diagram of the TALE and dCas9/gRNA targeting sites at the mouse Oct4 and Nanog enhancers. Red arrows indicate the gRNA targeting sites and the blue arrows mark the TALE sites. ( C ) Activation of the Oct4 distal enhancer luciferase reporter by the TALE-As and dCas9-As/gRNAs. ( D ) qRT-PCR analysis of the Oct4 mRNA levels in MEFs expressing the TALE-As or dCas9-As/gRNAs. ( E ) Activation of the Nanog enhancer luciferase reporter by the TALE-As and dCas9-As/gRNAs. ( F ) qRT-PCR analysis of the Nanog mRNA levels in EpiSCs expressing the TALE-As or dCas9-As/gRNAs. All gene expression values were normalized to Gapdh . Results were representative of three independent experiments and were presented as ±SD, n = 3. * P
    Shrna Expression Cassette, supplied by Addgene inc, used in various techniques. Bioz Stars score: 89/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    M6P/IGF2R expression increases during monocyte differentiation to macrophages . ( A ) Cell‐surface expression of M6P/IGF2R on isolated PBMC and MACS‐enriched monocytes from healthy donors was evaluated with mAb MEM‐238‐AF647 and flow cytometry. In parallel, MOPC‐21‐AF647 was used as an isotype control mAb, displayed by the cut‐off gates. The same analysis was performed with macrophages differentiated from MACS‐sorted monocytes during a 7‐day culture with recombinant human M‐CSF (50 ng/ml) followed by 2 days resting in macrophage serum free medium. ( B ) Primary human monocytes and monocyte‐derived macrophages from (A) were lysed and RNA was extracted. cDNA was synthesized from total RNA and gene expression was measured by real‐time PCR as described in the Material and Methods section with TaqMan primer sets for human M6P/IGF2R and YWHAZ as endogenous control. The M6P/IGF2R mean expression values relative to that of monocytes ± SD from 3 donors is shown

    Journal: Journal of Leukocyte Biology

    Article Title: The mannose 6‐phosphate/insulin‐like growth factor 2 receptor mediates plasminogen‐induced efferocytosis, et al. The mannose 6‐phosphate/insulin‐like growth factor 2 receptor mediates plasminogen‐induced efferocytosis

    doi: 10.1002/JLB.1AB0417-160RR

    Figure Lengend Snippet: M6P/IGF2R expression increases during monocyte differentiation to macrophages . ( A ) Cell‐surface expression of M6P/IGF2R on isolated PBMC and MACS‐enriched monocytes from healthy donors was evaluated with mAb MEM‐238‐AF647 and flow cytometry. In parallel, MOPC‐21‐AF647 was used as an isotype control mAb, displayed by the cut‐off gates. The same analysis was performed with macrophages differentiated from MACS‐sorted monocytes during a 7‐day culture with recombinant human M‐CSF (50 ng/ml) followed by 2 days resting in macrophage serum free medium. ( B ) Primary human monocytes and monocyte‐derived macrophages from (A) were lysed and RNA was extracted. cDNA was synthesized from total RNA and gene expression was measured by real‐time PCR as described in the Material and Methods section with TaqMan primer sets for human M6P/IGF2R and YWHAZ as endogenous control. The M6P/IGF2R mean expression values relative to that of monocytes ± SD from 3 donors is shown

    Article Snippet: 2.5 RNA interference, ZFN, and retroviral transduction technology The stable knockdown of M6P/IGF2R in THP‐1 by RNAi was generated by the delivery of a short hairpin RNA expression cassette as described in detail elsewhere., Accordingly, we applied 2 constructs targeting M6P/IGF2R mRNA, i.e., at the position 6588 (shM6P/IGF2R‐1) and 4525 (shM6P/IGF2R‐2)., As a control construct, the MISSION nontarget shRNA control vector (pLKO.1 puro) from Sigma–Aldrich was applied.

    Techniques: Expressing, Isolation, Magnetic Cell Separation, Flow Cytometry, Cytometry, Recombinant, Derivative Assay, Synthesized, Real-time Polymerase Chain Reaction

    Typical experimental workflow for GO-CRISPR screening and analysis using TRACS. (A) iOvCa147 High-grade serous ovarian cancer (HGSOC) cells were transduced with lentivirus expressing Cas9. High efficiency Cas9-positive cells (top row) and Cas9-negative cells (bottom row) were transduced with the GeCKO v2 pooled sgRNA library (L 0 ). After antibiotic selection, both Cas9 positive and negative cells were split into triplicates (x3) and maintained in initial culture conditions (T 0 ) before being transferred to suspension culture conditions in ULA plasticware (selective pressure, P s ) to induce spheroid formation and select for cell survival. Viable spheroid cells were then transferred to standard plasticware to facilitate reattachment in the final culture condition (T f ). The initial pooled sgRNA library (L 0 ) and Cas9-positive and Cas9-negative cells were collected at T 0 and T f for sgRNA quantitation by NGS. TRACS was used to calculate Library, Initial and Final Enrichment Scores (ES) using read quantities from L 0 and Cas9-positive and Cas9-negative samples. (B) 3D plot output from TRACS illustrating the Library ES, Initial ES and Final ES for each gene. Genes highlighted in dark blue have low Library ES (determined by calculating the first quartile value across all Library ES;

    Journal: bioRxiv

    Article Title: GO-CRISPR: a highly controlled workflow to discover gene essentiality in loss-of-function screens

    doi: 10.1101/2020.06.04.134841

    Figure Lengend Snippet: Typical experimental workflow for GO-CRISPR screening and analysis using TRACS. (A) iOvCa147 High-grade serous ovarian cancer (HGSOC) cells were transduced with lentivirus expressing Cas9. High efficiency Cas9-positive cells (top row) and Cas9-negative cells (bottom row) were transduced with the GeCKO v2 pooled sgRNA library (L 0 ). After antibiotic selection, both Cas9 positive and negative cells were split into triplicates (x3) and maintained in initial culture conditions (T 0 ) before being transferred to suspension culture conditions in ULA plasticware (selective pressure, P s ) to induce spheroid formation and select for cell survival. Viable spheroid cells were then transferred to standard plasticware to facilitate reattachment in the final culture condition (T f ). The initial pooled sgRNA library (L 0 ) and Cas9-positive and Cas9-negative cells were collected at T 0 and T f for sgRNA quantitation by NGS. TRACS was used to calculate Library, Initial and Final Enrichment Scores (ES) using read quantities from L 0 and Cas9-positive and Cas9-negative samples. (B) 3D plot output from TRACS illustrating the Library ES, Initial ES and Final ES for each gene. Genes highlighted in dark blue have low Library ES (determined by calculating the first quartile value across all Library ES;

    Article Snippet: They were transduced with viral particles encoding a Cas9 expression cassette (lentiCas9-Blast, Addgene #52962) to generate cells constitutively expressing Cas9 (Cas9-positive cells).

    Techniques: CRISPR, Transduction, Expressing, Selection, Quantitation Assay, Next-Generation Sequencing

    Validation of TRACS gene essentiality predictions. (A) The GeCKO v2 pooled library contains 1,000 non-targeting control (NTC) sgRNAs that should not elicit a change in cell fitness. We evaluated the ability of TRACS to classify these sgRNAs by computing a receiver operating characteristic curve (ROC). The area under the curve (AUC) was determined to be 98.5%, indicating TRACS accurately classifies these NTC sgRNAs as non-essential. (B) We evaluated the essentiality of the top five genes with the most negative ER: AGPS, SLC2A11, ZC3H7A, PDCD2L, NPM1 (see Table 1 ). CRISPR/Cas9 was used to disrupt each gene in iOvCa147 cells and pure single-gene knockout populations were assayed for spheroid cell viability in suspension culture conditions. Disruption of these genes resulted in significantly reduced cell viability. Genes in bar graph are arranged from most negative ER to least negative ER. (C) We similarly knocked out the top five genes with the most positive ER ( EPS15, hsa-mir-761, RPAP1, SYAP1, TRAF3IP1 ) and assayed for viability in suspension culture conditions. Disruption of these genes did not adversely affect viability. Genes in bar graph are arranged from smallest to largest ER. For B and C, each point represents a biological replicate (n = 6). Bars represent means and error bars represent standard deviation. Statistics was performed using one-way ANOVA; ** denotes p

    Journal: bioRxiv

    Article Title: GO-CRISPR: a highly controlled workflow to discover gene essentiality in loss-of-function screens

    doi: 10.1101/2020.06.04.134841

    Figure Lengend Snippet: Validation of TRACS gene essentiality predictions. (A) The GeCKO v2 pooled library contains 1,000 non-targeting control (NTC) sgRNAs that should not elicit a change in cell fitness. We evaluated the ability of TRACS to classify these sgRNAs by computing a receiver operating characteristic curve (ROC). The area under the curve (AUC) was determined to be 98.5%, indicating TRACS accurately classifies these NTC sgRNAs as non-essential. (B) We evaluated the essentiality of the top five genes with the most negative ER: AGPS, SLC2A11, ZC3H7A, PDCD2L, NPM1 (see Table 1 ). CRISPR/Cas9 was used to disrupt each gene in iOvCa147 cells and pure single-gene knockout populations were assayed for spheroid cell viability in suspension culture conditions. Disruption of these genes resulted in significantly reduced cell viability. Genes in bar graph are arranged from most negative ER to least negative ER. (C) We similarly knocked out the top five genes with the most positive ER ( EPS15, hsa-mir-761, RPAP1, SYAP1, TRAF3IP1 ) and assayed for viability in suspension culture conditions. Disruption of these genes did not adversely affect viability. Genes in bar graph are arranged from smallest to largest ER. For B and C, each point represents a biological replicate (n = 6). Bars represent means and error bars represent standard deviation. Statistics was performed using one-way ANOVA; ** denotes p

    Article Snippet: They were transduced with viral particles encoding a Cas9 expression cassette (lentiCas9-Blast, Addgene #52962) to generate cells constitutively expressing Cas9 (Cas9-positive cells).

    Techniques: CRISPR, Gene Knockout, Standard Deviation

    Enhancement of knock-in efficiency with the LoAD system. a Schematic of conventional overexpression and the LoAD system. Effector proteins such as CtIP can accumulate at the DSB site via the interaction between MS2 RNA loop and MS2 coat protein, resulting in programmable genome editing. sgRNA MS2 , sgRNA with two MS2 RNA loops. b Schematic of PITCh-mediated knock-in of fluorescent protein gene, harnessing MMEJ for the predefined, seamless gene cassette integration. This schematic only shows gene targeting in the CANX locus, but other gene loci such as ATB5B , PARP1 , FBL , and RPL11 were targeted in a similar way. c Knock-in frequency at the CANX locus measured by FACS analysis. The recruitment of MS2-CtIP with the LoAD system resulted in about 50% gene knock-in without any selection. Data are expressed as means ± s.e.m. ( n = 3). ** P

    Journal: Nature Communications

    Article Title: Biased genome editing using the local accumulation of DSB repair molecules system

    doi: 10.1038/s41467-018-05773-6

    Figure Lengend Snippet: Enhancement of knock-in efficiency with the LoAD system. a Schematic of conventional overexpression and the LoAD system. Effector proteins such as CtIP can accumulate at the DSB site via the interaction between MS2 RNA loop and MS2 coat protein, resulting in programmable genome editing. sgRNA MS2 , sgRNA with two MS2 RNA loops. b Schematic of PITCh-mediated knock-in of fluorescent protein gene, harnessing MMEJ for the predefined, seamless gene cassette integration. This schematic only shows gene targeting in the CANX locus, but other gene loci such as ATB5B , PARP1 , FBL , and RPL11 were targeted in a similar way. c Knock-in frequency at the CANX locus measured by FACS analysis. The recruitment of MS2-CtIP with the LoAD system resulted in about 50% gene knock-in without any selection. Data are expressed as means ± s.e.m. ( n = 3). ** P

    Article Snippet: For the addition of MS2 stem loops to sgRNA scaffold, the sgRNA expression cassette of the pX330A and pX330S vectors was replaced with that of the sgRNA(MS2) cloning backbone vector (Plasmid #61424, Addgene).

    Techniques: Knock-In, Over Expression, FACS, Gene Knock-In, Selection

    Domain insertion into AcrIIC1 yields a highly potent Nme Cas9 inhibitor. ( a , b ) HEK 293T cells were co-transfected with vectors expressing Nme Cas9, the indicated Acr and sgRNAs targeting different genomic loci followed by T7 endonuclease assay. In a , Acr: Nme Cas9 vector ratio used during transfection was 1:1, while in b , the indicated, low Acr: Nme Cas9 vector ratios were used. Representative T7 gel images and corresponding quantification of indel frequencies are shown. Lines in plots show means, dots are individual data points for n = 4 (DHFR, AAVS1 and IL2RG locus) or n = 3 (F8 locus) independent experiments. Chim., AcrIIC1-mCherry chimeras in Supplementary Fig. 2. In., input band. T7, T7 cleavage fragments. ( c ) AcrIIC1-mCherry chimeras outperform wild-type AcrIIC1 and AcrIIC3. Cells were co-transfected with vectors encoding Nme Cas9, a firefly luciferase reporter and corresponding reporter-targeting sgRNA as well as the indicated Acr followed by luciferase assay. Bars indicate means, error bars the SD for n = 3 independent experiments. ( a-c ) N, negative (Cas 9 only ( a , b ) or reporter only ( c ) control (Ctrl.)). P, positive control (Cas9 + sgRNA ( a , b ) or reporter + Cas9 ( c )). * P

    Journal: bioRxiv

    Article Title: Computational design of anti-CRISPR proteins with improved inhibition potency and expanded specificity

    doi: 10.1101/685032

    Figure Lengend Snippet: Domain insertion into AcrIIC1 yields a highly potent Nme Cas9 inhibitor. ( a , b ) HEK 293T cells were co-transfected with vectors expressing Nme Cas9, the indicated Acr and sgRNAs targeting different genomic loci followed by T7 endonuclease assay. In a , Acr: Nme Cas9 vector ratio used during transfection was 1:1, while in b , the indicated, low Acr: Nme Cas9 vector ratios were used. Representative T7 gel images and corresponding quantification of indel frequencies are shown. Lines in plots show means, dots are individual data points for n = 4 (DHFR, AAVS1 and IL2RG locus) or n = 3 (F8 locus) independent experiments. Chim., AcrIIC1-mCherry chimeras in Supplementary Fig. 2. In., input band. T7, T7 cleavage fragments. ( c ) AcrIIC1-mCherry chimeras outperform wild-type AcrIIC1 and AcrIIC3. Cells were co-transfected with vectors encoding Nme Cas9, a firefly luciferase reporter and corresponding reporter-targeting sgRNA as well as the indicated Acr followed by luciferase assay. Bars indicate means, error bars the SD for n = 3 independent experiments. ( a-c ) N, negative (Cas 9 only ( a , b ) or reporter only ( c ) control (Ctrl.)). P, positive control (Cas9 + sgRNA ( a , b ) or reporter + Cas9 ( c )). * P

    Article Snippet: The plasmid pEJS654 All-in-One AAV-sgRNA-hNmeCas9 co-encoding Nme Cas9 and a corresponding sgRNA expression cassette was a kind gift from Erik Sontheimer (Addgene #112139).

    Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Positive Control

    No effects of CatD upregulation on HK2 proliferation under culture with normal glucose. (A) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h, then cultured for additional 48 h. Transfection efficiency was analyzed by measurement of GFP+ positive cells under a fluorescence microscopy, and representative images are provided. (B) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h, western blot was performed using the indicated antibodies. (C) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h. Viable cells were counted using trypan blue exclusion experiment daily. (D) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h. Cell proliferation was analyzed using RTCA in a real-time pattern. (E) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h. DNA synthesis was analyzed using the EdU incorporation assay, and representative images are provided. (F) Total and EdU-positive cells in (E) were counted, and the rate of EdU incorporation was calculated. (G) HK2 cells were infected with lentiviral vectors containing empty or CatD construct, and cell cycle progression was analyzed using flowcytometry. N.S., not significant.

    Journal: American Journal of Translational Research

    Article Title: Cathepsin D protects renal tubular cells from damage induced by high glucose independent of its enzymatic activity

    doi:

    Figure Lengend Snippet: No effects of CatD upregulation on HK2 proliferation under culture with normal glucose. (A) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h, then cultured for additional 48 h. Transfection efficiency was analyzed by measurement of GFP+ positive cells under a fluorescence microscopy, and representative images are provided. (B) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h, western blot was performed using the indicated antibodies. (C) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h. Viable cells were counted using trypan blue exclusion experiment daily. (D) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h. Cell proliferation was analyzed using RTCA in a real-time pattern. (E) HK2 cells were infected with lentiviral vectors containing empty or CatD construct for 12 h and cultured for additional 48 h. DNA synthesis was analyzed using the EdU incorporation assay, and representative images are provided. (F) Total and EdU-positive cells in (E) were counted, and the rate of EdU incorporation was calculated. (G) HK2 cells were infected with lentiviral vectors containing empty or CatD construct, and cell cycle progression was analyzed using flowcytometry. N.S., not significant.

    Article Snippet: Lentiviral plasmids containing CatD cDNA and a GFP expression cassette were produced by GeneChem Corporation (Shanghai, China).

    Techniques: Infection, Construct, Cell Culture, Transfection, Fluorescence, Microscopy, Western Blot, DNA Synthesis

    Effect of shRNAs 1 and 2 on MCMV replication at m .o.i. = 0.2. a) An SEC expressing shRNA-2 was transfected into M2-10B4 cells 24 hrs prior to infection with MCMV at an m.o.i. of 0.2. b) Western blot of IE-3 protein levels in shRNA-2 treated and control shRNA treated samples Effect of MCMV specific shRNA at low m.o.i.'s in vitro . c) Virus titers at day 5 p.i. in M2-10B4 cultures infected with MCMV at the indicated m.o.i.'s. Dark bars: 50 nM IE-3 specific shRNA-1, white bars: control shRNA. d) Western blot showing the time course of IE-3 protein expression in MCMV-infected M2-10B4 cells (m.o.i. = 0.2) following transfection of the IE-3 shRNA-1 expressing plasmid. e) Viral titers at day 5 p.i. in M2-10B4 cultures infected with MCMV at various m.o.i.'s and treated with either shRNA-1a or shRNA-2a.

    Journal: Herpesviridae

    Article Title: The effect of murine cytomegalovirus IE-3 specific shRNA is dependent on intragenic target site due to multiple transcription initiation sites

    doi: 10.1186/2042-4280-2-9

    Figure Lengend Snippet: Effect of shRNAs 1 and 2 on MCMV replication at m .o.i. = 0.2. a) An SEC expressing shRNA-2 was transfected into M2-10B4 cells 24 hrs prior to infection with MCMV at an m.o.i. of 0.2. b) Western blot of IE-3 protein levels in shRNA-2 treated and control shRNA treated samples Effect of MCMV specific shRNA at low m.o.i.'s in vitro . c) Virus titers at day 5 p.i. in M2-10B4 cultures infected with MCMV at the indicated m.o.i.'s. Dark bars: 50 nM IE-3 specific shRNA-1, white bars: control shRNA. d) Western blot showing the time course of IE-3 protein expression in MCMV-infected M2-10B4 cells (m.o.i. = 0.2) following transfection of the IE-3 shRNA-1 expressing plasmid. e) Viral titers at day 5 p.i. in M2-10B4 cultures infected with MCMV at various m.o.i.'s and treated with either shRNA-1a or shRNA-2a.

    Article Snippet: shRNAs and plasmids shRNAs were transcribed from an shRNA expression cassette (SEC) intracellularly under the control of a mouse U6 promoter following cloning of a hairpin producing cDNA into an SEC vector using the Silencer Express siRNA Expression Cassette Kit (Ambion Inc., Austin, Texas).

    Techniques: Size-exclusion Chromatography, Expressing, shRNA, Transfection, Infection, Western Blot, In Vitro, Plasmid Preparation

    Construction and analysis of pHSVF-CREin. a The infectious clone pHSVF-CREin was made by insertion of the CREin expression cassette into the pHSVF-BFP infectious clone in a process paralleling that described in Fig. 1 . The CREin expression cassette was PCR amplified from the pEP-CREin-in template and recombined into pHSVF-BFP BAC vector backbone by lambda RED recombination, resulting in the replacement of the TagBFP coding sequence with that of CREin::kanamycin. In the second recombination step, the kanamycin resistance gene (aphA1) was removed based on partially duplicated sequences in the flanking CREin coding sequence ( red boxes ), which simultaneously established the contiguous CREin coding sequence and resulted in pHSVF-CREin. Excision of the BAC from pHSVF-CREin was achieved by autonomous expression of Cre recombinase following introduction of the DNA into either Vero or HEK293T cells, resulting in a passage 1 (P1) harvest. b MluI ( left ) or PvuI ( right ) restriction analysis of HSV-1 DNA harvested from purified nucleocapsids. Yellow arrow heads indicate restriction fragments that mark the presence or absence of the BAC vector sequences. Size standards are indicated in kb. c Amplification of viral and plasmid DNA using primers designed to detect removal of BAC DNA from the viral genome. The positions of the primer pairs are indicated in panel A

    Journal: BMC Biotechnology

    Article Title: New tools to convert bacterial artificial chromosomes to a self-excising design and their application to a herpes simplex virus type 1 infectious clone

    doi: 10.1186/s12896-016-0295-4

    Figure Lengend Snippet: Construction and analysis of pHSVF-CREin. a The infectious clone pHSVF-CREin was made by insertion of the CREin expression cassette into the pHSVF-BFP infectious clone in a process paralleling that described in Fig. 1 . The CREin expression cassette was PCR amplified from the pEP-CREin-in template and recombined into pHSVF-BFP BAC vector backbone by lambda RED recombination, resulting in the replacement of the TagBFP coding sequence with that of CREin::kanamycin. In the second recombination step, the kanamycin resistance gene (aphA1) was removed based on partially duplicated sequences in the flanking CREin coding sequence ( red boxes ), which simultaneously established the contiguous CREin coding sequence and resulted in pHSVF-CREin. Excision of the BAC from pHSVF-CREin was achieved by autonomous expression of Cre recombinase following introduction of the DNA into either Vero or HEK293T cells, resulting in a passage 1 (P1) harvest. b MluI ( left ) or PvuI ( right ) restriction analysis of HSV-1 DNA harvested from purified nucleocapsids. Yellow arrow heads indicate restriction fragments that mark the presence or absence of the BAC vector sequences. Size standards are indicated in kb. c Amplification of viral and plasmid DNA using primers designed to detect removal of BAC DNA from the viral genome. The positions of the primer pairs are indicated in panel A

    Article Snippet: The resulting HSV-1 infectious clone contained the TagBFP expression cassette in the BAC vector backbone (see Fig. ).

    Techniques: Expressing, Polymerase Chain Reaction, Amplification, BAC Assay, Plasmid Preparation, Sequencing, Purification

    Construction and analysis of pHSVF-BFP. a Flow diagram of two-step recombination (En Passant) used to insert a TagBFP expression cassette into the vector backbone of pYEbac102 in E. coli , and subsequent Cre-based removal of the BAC vector from the viral genome in mammalian cells. The expression cassette was PCR amplified from the pEP-TagBFP-in template and recombined into the BAC vector backbone by lambda RED recombination using kanamycin resistance as selective pressure. In a second recombination step, the kanamycin resistance gene (aphA1) was removed based on partially duplicated sequences in the flanking TagBFP coding sequence ( red boxes ), which simultaneously established the contiguous BFP coding sequence and resulted in pHSVF-BFP. Transfection of pHSVF-BFP into Vero cells produced the vHSVF-BFP virus that stably expressed blue fluorescence, which was further expanded by a second passage (P2) on Vero cells. Transfection and serial passage in Vero-cre cells produced HSV-1 lacking the BAC backbone and associated fluorescence but retaining a single loxP site ( black circles ) between the UL3 and UL4 genes. b Excision of the pBeloBAC vector from vHSVF-BFP was monitored by fluorescent plaque assay. Following transfection of Vero cells (P1), vHSVF-BFP was successively passaged on Vero-cre cells for a second (P2cre), third (P3cre), and fourth round (P4cre), or on Vero cells that did not express Cre recombinase for a second round (P2). Plaques produced from each harvest were scored as positive ( blue ) or negative ( black ) for fluorescence. The data are a composite of three independent experiments consisting of > 40 plaques scored per experiment. Error bars are SD

    Journal: BMC Biotechnology

    Article Title: New tools to convert bacterial artificial chromosomes to a self-excising design and their application to a herpes simplex virus type 1 infectious clone

    doi: 10.1186/s12896-016-0295-4

    Figure Lengend Snippet: Construction and analysis of pHSVF-BFP. a Flow diagram of two-step recombination (En Passant) used to insert a TagBFP expression cassette into the vector backbone of pYEbac102 in E. coli , and subsequent Cre-based removal of the BAC vector from the viral genome in mammalian cells. The expression cassette was PCR amplified from the pEP-TagBFP-in template and recombined into the BAC vector backbone by lambda RED recombination using kanamycin resistance as selective pressure. In a second recombination step, the kanamycin resistance gene (aphA1) was removed based on partially duplicated sequences in the flanking TagBFP coding sequence ( red boxes ), which simultaneously established the contiguous BFP coding sequence and resulted in pHSVF-BFP. Transfection of pHSVF-BFP into Vero cells produced the vHSVF-BFP virus that stably expressed blue fluorescence, which was further expanded by a second passage (P2) on Vero cells. Transfection and serial passage in Vero-cre cells produced HSV-1 lacking the BAC backbone and associated fluorescence but retaining a single loxP site ( black circles ) between the UL3 and UL4 genes. b Excision of the pBeloBAC vector from vHSVF-BFP was monitored by fluorescent plaque assay. Following transfection of Vero cells (P1), vHSVF-BFP was successively passaged on Vero-cre cells for a second (P2cre), third (P3cre), and fourth round (P4cre), or on Vero cells that did not express Cre recombinase for a second round (P2). Plaques produced from each harvest were scored as positive ( blue ) or negative ( black ) for fluorescence. The data are a composite of three independent experiments consisting of > 40 plaques scored per experiment. Error bars are SD

    Article Snippet: The resulting HSV-1 infectious clone contained the TagBFP expression cassette in the BAC vector backbone (see Fig. ).

    Techniques: Flow Cytometry, Expressing, Plasmid Preparation, BAC Assay, Polymerase Chain Reaction, Amplification, Sequencing, Transfection, Produced, Stable Transfection, Fluorescence, Plaque Assay

    In vivo dCas9 association assay for anti-CRISPR proteins. (a) A yeast strain was constructed harbouring an inducible dCas9 (D10A H840A) fused to both mCherry and LactC2 at its C-terminus (GFY-3104) and transformed with (i) a sgRNA[u1] plasmid (pGF-V1220) and (ii) GFP-tagged AcrIIA2/A4-containing plasmids (pGF-IVL1384 to pGF-IVL1387). Strains were cultured overnight in raffinose/sucrose medium lacking uracil and lacking leucine, back-diluted into synthetic medium containing galactose also lacking uracil and leucine, and grown for 4.5 h at 30 °C. Cells were harvested, washed with water and imaged by fluorescence microscopy. Representative images are displayed. Scale bar, 3 µm. (b) A measure of the ratio between the maximum pixel intensity located on the PM was compared to a sampling of the average cytosolic pixel intensity for the GFP signal ( n =15–30 cells per genotype). Ten random individual measurements were taken for both the PM and cytosolic levels per cell. Error, sd . Three additional AcrIIA4 constructs were tested (asterisks) containing sets of two alanine substitutions (pGF-IVL1431 to pGF-IVL1433). Bottom , select strains were compared using an unpaired t -test. Red text, P -values greater than 0.05.

    Journal: Microbiology

    Article Title: Gene drive inhibition by the anti-CRISPR proteins AcrIIA2 and AcrIIA4 in Saccharomyces cerevisiae

    doi: 10.1099/mic.0.000635

    Figure Lengend Snippet: In vivo dCas9 association assay for anti-CRISPR proteins. (a) A yeast strain was constructed harbouring an inducible dCas9 (D10A H840A) fused to both mCherry and LactC2 at its C-terminus (GFY-3104) and transformed with (i) a sgRNA[u1] plasmid (pGF-V1220) and (ii) GFP-tagged AcrIIA2/A4-containing plasmids (pGF-IVL1384 to pGF-IVL1387). Strains were cultured overnight in raffinose/sucrose medium lacking uracil and lacking leucine, back-diluted into synthetic medium containing galactose also lacking uracil and leucine, and grown for 4.5 h at 30 °C. Cells were harvested, washed with water and imaged by fluorescence microscopy. Representative images are displayed. Scale bar, 3 µm. (b) A measure of the ratio between the maximum pixel intensity located on the PM was compared to a sampling of the average cytosolic pixel intensity for the GFP signal ( n =15–30 cells per genotype). Ten random individual measurements were taken for both the PM and cytosolic levels per cell. Error, sd . Three additional AcrIIA4 constructs were tested (asterisks) containing sets of two alanine substitutions (pGF-IVL1431 to pGF-IVL1433). Bottom , select strains were compared using an unpaired t -test. Red text, P -values greater than 0.05.

    Article Snippet: For all substitutions, the AcrIIA2 and AcrIIA4 expression cassettes were amplified, cloned into a TOPO II vector (pCR-Blunt II-TOPO, Invitrogen), mutagenized by PCR, and sub-cloned to pRS316 using flanking NotI/SpeI restriction sites.

    Techniques: In Vivo, Histone Association Assay, CRISPR, Construct, Transformation Assay, Plasmid Preparation, Cell Culture, Fluorescence, Microscopy, Sampling

    Mutational analysis of the Cas9 inhibitory function of AcrIIA2 in an active gene drive system. (a) Vectors expressing AcrIIA2 mutants ( Fig. 3 , top left , red text) and the sgRNA[u1] plasmid (pGF-V1220) were transformed into yeast along with controls (2x, empty pRS316, pRS425; 1x, empty pRS316, sgRNA[u1] plasmid). Gene drive strains were mated to the target strains (GFY-3206 and GFY-3207) and the diploid strains were induced in rich media containing galactose for 5 h before plating. The percentage of colonies displaying an active drive system was quantified in duplicate. Error, sd . (b) Vectors expressing AcrIIA4 mutants ( Fig. 3 , top right , red text) and the sgRNA[u1] plasmid were analysed as in (a). Additional mutants not previously tested were also included (pGF-V1470 to pGF-V1485 and pGF-V1534 to pGF-V1536). Blue asterisks, residues tested by previous groups [ 25, 26 ] shown to contribute to AcrIIA4 association with sgRNA-loaded Cas9. Red asterisks, mutational substitutions displaying a partial loss of inhibitory function not previously documented. (c) Left, crystal structure of the AcrIIA4 protein bound to Cas9/sgRNA (PDB 5XBL). Cyan-labelled residues (and side chains) are residues previously demonstrated to be critical for AcrIIA4 association with Cas9. Yellow-labelled residues (red text) illustrate three substitutions found to partially reduce AcrIIA4 inhibitory function in vivo. Right, side views of AcrIIA4 with a 180° rotation.

    Journal: Microbiology

    Article Title: Gene drive inhibition by the anti-CRISPR proteins AcrIIA2 and AcrIIA4 in Saccharomyces cerevisiae

    doi: 10.1099/mic.0.000635

    Figure Lengend Snippet: Mutational analysis of the Cas9 inhibitory function of AcrIIA2 in an active gene drive system. (a) Vectors expressing AcrIIA2 mutants ( Fig. 3 , top left , red text) and the sgRNA[u1] plasmid (pGF-V1220) were transformed into yeast along with controls (2x, empty pRS316, pRS425; 1x, empty pRS316, sgRNA[u1] plasmid). Gene drive strains were mated to the target strains (GFY-3206 and GFY-3207) and the diploid strains were induced in rich media containing galactose for 5 h before plating. The percentage of colonies displaying an active drive system was quantified in duplicate. Error, sd . (b) Vectors expressing AcrIIA4 mutants ( Fig. 3 , top right , red text) and the sgRNA[u1] plasmid were analysed as in (a). Additional mutants not previously tested were also included (pGF-V1470 to pGF-V1485 and pGF-V1534 to pGF-V1536). Blue asterisks, residues tested by previous groups [ 25, 26 ] shown to contribute to AcrIIA4 association with sgRNA-loaded Cas9. Red asterisks, mutational substitutions displaying a partial loss of inhibitory function not previously documented. (c) Left, crystal structure of the AcrIIA4 protein bound to Cas9/sgRNA (PDB 5XBL). Cyan-labelled residues (and side chains) are residues previously demonstrated to be critical for AcrIIA4 association with Cas9. Yellow-labelled residues (red text) illustrate three substitutions found to partially reduce AcrIIA4 inhibitory function in vivo. Right, side views of AcrIIA4 with a 180° rotation.

    Article Snippet: For all substitutions, the AcrIIA2 and AcrIIA4 expression cassettes were amplified, cloned into a TOPO II vector (pCR-Blunt II-TOPO, Invitrogen), mutagenized by PCR, and sub-cloned to pRS316 using flanking NotI/SpeI restriction sites.

    Techniques: Expressing, Plasmid Preparation, Transformation Assay, In Vivo

    Inhibition of S. pyogenes Cas9 editing through in vivo expression of AcrIIA2 and AcrIIA4. (a) The anti-CRISPR genes AcrIIA2 and AcrIIA4 were cloned (pGF-IVL1384 to pGF-IVL1387) under control of the CDC11 promoter on CEN -based plasmids, tagged with GFP at either their N- or C-terminus, transformed into WT yeast (BY4741) and imaged by fluorescence microscopy. White dotted outline, cell periphery. Scale bar, 3 µm. Representative cells are illustrated ( left ). An average pixel intensity (cytosol) was measured for individual cells ( right, n =30–75 cells per genotype). Error, sd . (b) The haploid yeast strain harbouring S. pyogenes Cas9 from Fig. 1 (GFY-2383) was first transformed with plasmids expressing either GFP-tagged (a) or untagged (pGF-IVL1336 and pGF-IVL1337) AcrIIA2 and AcrIIA4 constructs. Second, following induction of Cas9 in galactose, yeasts were transformed with equimolar amounts of sgRNA[u2]-containing plasmid (pGF-V809), and the total number of colonies was quantified on SD-URA-LEU plates in triplicate. Error, sd . Select comparisons between experimental conditions ( left ) were analysed using an unpaired t -test. Red text highlights P -values greater than 0.05.

    Journal: Microbiology

    Article Title: Gene drive inhibition by the anti-CRISPR proteins AcrIIA2 and AcrIIA4 in Saccharomyces cerevisiae

    doi: 10.1099/mic.0.000635

    Figure Lengend Snippet: Inhibition of S. pyogenes Cas9 editing through in vivo expression of AcrIIA2 and AcrIIA4. (a) The anti-CRISPR genes AcrIIA2 and AcrIIA4 were cloned (pGF-IVL1384 to pGF-IVL1387) under control of the CDC11 promoter on CEN -based plasmids, tagged with GFP at either their N- or C-terminus, transformed into WT yeast (BY4741) and imaged by fluorescence microscopy. White dotted outline, cell periphery. Scale bar, 3 µm. Representative cells are illustrated ( left ). An average pixel intensity (cytosol) was measured for individual cells ( right, n =30–75 cells per genotype). Error, sd . (b) The haploid yeast strain harbouring S. pyogenes Cas9 from Fig. 1 (GFY-2383) was first transformed with plasmids expressing either GFP-tagged (a) or untagged (pGF-IVL1336 and pGF-IVL1337) AcrIIA2 and AcrIIA4 constructs. Second, following induction of Cas9 in galactose, yeasts were transformed with equimolar amounts of sgRNA[u2]-containing plasmid (pGF-V809), and the total number of colonies was quantified on SD-URA-LEU plates in triplicate. Error, sd . Select comparisons between experimental conditions ( left ) were analysed using an unpaired t -test. Red text highlights P -values greater than 0.05.

    Article Snippet: For all substitutions, the AcrIIA2 and AcrIIA4 expression cassettes were amplified, cloned into a TOPO II vector (pCR-Blunt II-TOPO, Invitrogen), mutagenized by PCR, and sub-cloned to pRS316 using flanking NotI/SpeI restriction sites.

    Techniques: Inhibition, In Vivo, Expressing, CRISPR, Clone Assay, Transformation Assay, Fluorescence, Microscopy, Construct, Plasmid Preparation

    Unbiased alanine scan of the AcrIIA2 and AcrIIA4 proteins and effects on inhibition of S. pyogenes Cas9 editing. (a) The AcrIIA2 protein was mutated using pairs of alanine substitutions – red text highlights all amino acids included in the mutation analysis ( top ). Yeast (GFY-2383) were first transformed with all URA3 -based plasmids: (i) empty pRS316, (ii) WT AcrIIA2 (pGF-IVL1336) or (iii) mutant AcrIIA2 (pGF-V1399 to pGF-V1420). Second, editing of haploid yeast was performed as previously described (see Methods). Briefly, following induction of Cas9, sgRNA[u2] plasmid (pGF-V809) was transformed, recovered overnight and plated to SD-URA-LEU medium. The total number of surviving colonies was quantified in triplicate. Error, sd . (b) A similar mutational analysis was performed on the A4 protein as in (a). Plasmids included: (i) empty pRS316, (ii) WT A4 (pGF-IVL1337) and (iii) mutant A4 (pGF-V1421 to pGF-V1439).

    Journal: Microbiology

    Article Title: Gene drive inhibition by the anti-CRISPR proteins AcrIIA2 and AcrIIA4 in Saccharomyces cerevisiae

    doi: 10.1099/mic.0.000635

    Figure Lengend Snippet: Unbiased alanine scan of the AcrIIA2 and AcrIIA4 proteins and effects on inhibition of S. pyogenes Cas9 editing. (a) The AcrIIA2 protein was mutated using pairs of alanine substitutions – red text highlights all amino acids included in the mutation analysis ( top ). Yeast (GFY-2383) were first transformed with all URA3 -based plasmids: (i) empty pRS316, (ii) WT AcrIIA2 (pGF-IVL1336) or (iii) mutant AcrIIA2 (pGF-V1399 to pGF-V1420). Second, editing of haploid yeast was performed as previously described (see Methods). Briefly, following induction of Cas9, sgRNA[u2] plasmid (pGF-V809) was transformed, recovered overnight and plated to SD-URA-LEU medium. The total number of surviving colonies was quantified in triplicate. Error, sd . (b) A similar mutational analysis was performed on the A4 protein as in (a). Plasmids included: (i) empty pRS316, (ii) WT A4 (pGF-IVL1337) and (iii) mutant A4 (pGF-V1421 to pGF-V1439).

    Article Snippet: For all substitutions, the AcrIIA2 and AcrIIA4 expression cassettes were amplified, cloned into a TOPO II vector (pCR-Blunt II-TOPO, Invitrogen), mutagenized by PCR, and sub-cloned to pRS316 using flanking NotI/SpeI restriction sites.

    Techniques: Inhibition, Mutagenesis, Transformation Assay, Plasmid Preparation

    AcrIIA2 and AcrIIA4 can inhibit CRISPR-based gene drives in yeast. (a) Design of an artificial gene drive system in S. cerevisiae . The haploid ‘gene drive’ strain (harbouring inducible S. pyogenes Cas9) was built in MATa yeast (GFY-2383) and included (i) a LEU2 -based high copy plasmid with the sgRNA[u1] cassette and (ii) the URA3 -based CEN plasmid expressing untagged AcrIIA2 or AcrIIA4 ( top ). Two nearly isogenic ‘target’ strains were built in MAT α yeast (GFY-3206 and GFY-3207) containing an artificial target gene and selectable HIS5 marker (from S. pombe ). The entire construct was flanked with two [u1] artificial target sites. Middle , three scenarios are depicted involving (i) a fully active gene drive, (ii) partially active drive activity and (iii) fully inhibited drive activity due to the presence of the anti-CRISPR proteins. (b) Activation of an artificial gene drive system in yeast. First, yeast were transformed with (i) either an empty vector (pRS316) or plasmid expressing AcrIIA2 (pGF-IVL1336) or AcrIIA4 (pGF-IVL1337) and (ii) the sgRNA[u2] plasmid (pGF-V1220), and mated to the target strains (pGF-3206 and pGF-3207) on rich medium (containing dextrose) for 24 h at 30 °C. Second, diploid yeast were obtained by velvet transfer of all colonies to SD-URA-LEU-HIS medium for two consecutive rounds of selection. Third, cultures of pre-induction medium (raffinose/sucrose lacking leucine and lacking uracil) were grown overnight, back-diluted into YPGal and cultured for between 0 and 12 h. Fourth, cells were harvested, washed and diluted to approximately 500–1000 cells per plate (SD-URA-LEU medium) and grown for 2–3 days. Fifth, yeasts were transferred by velvet to an identical plate type and SD-HIS medium for an additional 24 h incubation before imaging. Representative plates for each time point are illustrated. (c) The total number of surviving colonies was quantified for each plate type in duplicate. ‘Gene drive activity’ was illustrated as the proportion of sampled colonies ( n =100–300 colonies per plate) sensitive to the SD-HIS condition (e.g. 99 % of colonies present on SD-URA-LEU plate but absent on the SD-HIS plate corresponds to 99 % gene drive activity). (d) Molecular analysis of diploid yeast following gene drive activation. Clonal isolates were obtained from the 0 and 12 h time points (SD-URA-LEU plate), chromosomal DNA was purified and PCRs were performed on the diploid genomes. Primer combinations and the expected fragment sizes ( right ) are illustrated in the gene drive schematic (a). Four representative isolates from each genotype are illustrated – for the gene drive containing AcrIIA2, two isolates displaying no growth on SD-HIS were also tested (red asterisk).

    Journal: Microbiology

    Article Title: Gene drive inhibition by the anti-CRISPR proteins AcrIIA2 and AcrIIA4 in Saccharomyces cerevisiae

    doi: 10.1099/mic.0.000635

    Figure Lengend Snippet: AcrIIA2 and AcrIIA4 can inhibit CRISPR-based gene drives in yeast. (a) Design of an artificial gene drive system in S. cerevisiae . The haploid ‘gene drive’ strain (harbouring inducible S. pyogenes Cas9) was built in MATa yeast (GFY-2383) and included (i) a LEU2 -based high copy plasmid with the sgRNA[u1] cassette and (ii) the URA3 -based CEN plasmid expressing untagged AcrIIA2 or AcrIIA4 ( top ). Two nearly isogenic ‘target’ strains were built in MAT α yeast (GFY-3206 and GFY-3207) containing an artificial target gene and selectable HIS5 marker (from S. pombe ). The entire construct was flanked with two [u1] artificial target sites. Middle , three scenarios are depicted involving (i) a fully active gene drive, (ii) partially active drive activity and (iii) fully inhibited drive activity due to the presence of the anti-CRISPR proteins. (b) Activation of an artificial gene drive system in yeast. First, yeast were transformed with (i) either an empty vector (pRS316) or plasmid expressing AcrIIA2 (pGF-IVL1336) or AcrIIA4 (pGF-IVL1337) and (ii) the sgRNA[u2] plasmid (pGF-V1220), and mated to the target strains (pGF-3206 and pGF-3207) on rich medium (containing dextrose) for 24 h at 30 °C. Second, diploid yeast were obtained by velvet transfer of all colonies to SD-URA-LEU-HIS medium for two consecutive rounds of selection. Third, cultures of pre-induction medium (raffinose/sucrose lacking leucine and lacking uracil) were grown overnight, back-diluted into YPGal and cultured for between 0 and 12 h. Fourth, cells were harvested, washed and diluted to approximately 500–1000 cells per plate (SD-URA-LEU medium) and grown for 2–3 days. Fifth, yeasts were transferred by velvet to an identical plate type and SD-HIS medium for an additional 24 h incubation before imaging. Representative plates for each time point are illustrated. (c) The total number of surviving colonies was quantified for each plate type in duplicate. ‘Gene drive activity’ was illustrated as the proportion of sampled colonies ( n =100–300 colonies per plate) sensitive to the SD-HIS condition (e.g. 99 % of colonies present on SD-URA-LEU plate but absent on the SD-HIS plate corresponds to 99 % gene drive activity). (d) Molecular analysis of diploid yeast following gene drive activation. Clonal isolates were obtained from the 0 and 12 h time points (SD-URA-LEU plate), chromosomal DNA was purified and PCRs were performed on the diploid genomes. Primer combinations and the expected fragment sizes ( right ) are illustrated in the gene drive schematic (a). Four representative isolates from each genotype are illustrated – for the gene drive containing AcrIIA2, two isolates displaying no growth on SD-HIS were also tested (red asterisk).

    Article Snippet: For all substitutions, the AcrIIA2 and AcrIIA4 expression cassettes were amplified, cloned into a TOPO II vector (pCR-Blunt II-TOPO, Invitrogen), mutagenized by PCR, and sub-cloned to pRS316 using flanking NotI/SpeI restriction sites.

    Techniques: CRISPR, Plasmid Preparation, Expressing, Marker, Construct, Activity Assay, Activation Assay, Transformation Assay, Selection, Cell Culture, Incubation, Imaging, Purification

    Temporal control of anti-CRISPR expression can modulate the overall activity of a nuclease-based gene drive. (a) Schematic of the gene drive system harbouring an inducible AcrIIA2 or AcrIIA4 inhibitor within the cassette. Modifications to the previously tested gene drive ( Figs 4 and 5 ) included integration of a second inducible expression cassette ( MET25 promoter) proximal to the S. pyogenes gene drive system controlling expression of either AcrIIA2 or AcrIIA4. The sgRNA targeted the mCherry gene within the target strains. (b) The two gene drive strains (GFY-3285 and GFY-3287) were transformed with the sgRNA(mCh) plasmid (pGF-425+IVL1277), mated to the target strains (GFY-3206 and GFY-3207) and diploids were selected on SD-LEU-HIS plates (three consecutive times). Diploids were then cultured overnight in pre-induction medium (raffinose/sucrose lacking leucine and containing methionine). Five distinct growth conditions were tested (labelled 1–5) altering the order of either Cas9 induction, AcrIIA2/A4 induction or control conditions. Cas9 induction included culturing in medium containing galactose. Inhibition (asterisk) of Cas9 expression included use of the raffinose/sucrose mixture. Activation of AcrIIA2/A4 included culturing in medium lacking methionine (repression by addition of methionine). All culturing steps also lacked leucine to maintain the sgRNA(mCh) plasmid. For conditions (1) and (5), a media switch occurred – after 2 h, yeast were harvested, washed four times with water and resuspended in the new media type for an additional 2 h. All diploids were plated on SD-LEU medium as previously described (500–1000 cells per plate). (c) The percentage of drive activity was quantified in duplicate. Error, sd . Below , representative plates from selected conditions are illustrated. Right , select combinations of experimental conditions for AcrIIA2 and AcrIIA4 were compared using an unpaired t -test. Red text, P -value of greater than 0.05.

    Journal: Microbiology

    Article Title: Gene drive inhibition by the anti-CRISPR proteins AcrIIA2 and AcrIIA4 in Saccharomyces cerevisiae

    doi: 10.1099/mic.0.000635

    Figure Lengend Snippet: Temporal control of anti-CRISPR expression can modulate the overall activity of a nuclease-based gene drive. (a) Schematic of the gene drive system harbouring an inducible AcrIIA2 or AcrIIA4 inhibitor within the cassette. Modifications to the previously tested gene drive ( Figs 4 and 5 ) included integration of a second inducible expression cassette ( MET25 promoter) proximal to the S. pyogenes gene drive system controlling expression of either AcrIIA2 or AcrIIA4. The sgRNA targeted the mCherry gene within the target strains. (b) The two gene drive strains (GFY-3285 and GFY-3287) were transformed with the sgRNA(mCh) plasmid (pGF-425+IVL1277), mated to the target strains (GFY-3206 and GFY-3207) and diploids were selected on SD-LEU-HIS plates (three consecutive times). Diploids were then cultured overnight in pre-induction medium (raffinose/sucrose lacking leucine and containing methionine). Five distinct growth conditions were tested (labelled 1–5) altering the order of either Cas9 induction, AcrIIA2/A4 induction or control conditions. Cas9 induction included culturing in medium containing galactose. Inhibition (asterisk) of Cas9 expression included use of the raffinose/sucrose mixture. Activation of AcrIIA2/A4 included culturing in medium lacking methionine (repression by addition of methionine). All culturing steps also lacked leucine to maintain the sgRNA(mCh) plasmid. For conditions (1) and (5), a media switch occurred – after 2 h, yeast were harvested, washed four times with water and resuspended in the new media type for an additional 2 h. All diploids were plated on SD-LEU medium as previously described (500–1000 cells per plate). (c) The percentage of drive activity was quantified in duplicate. Error, sd . Below , representative plates from selected conditions are illustrated. Right , select combinations of experimental conditions for AcrIIA2 and AcrIIA4 were compared using an unpaired t -test. Red text, P -value of greater than 0.05.

    Article Snippet: For all substitutions, the AcrIIA2 and AcrIIA4 expression cassettes were amplified, cloned into a TOPO II vector (pCR-Blunt II-TOPO, Invitrogen), mutagenized by PCR, and sub-cloned to pRS316 using flanking NotI/SpeI restriction sites.

    Techniques: CRISPR, Expressing, Activity Assay, Transformation Assay, Plasmid Preparation, Cell Culture, Inhibition, Activation Assay

    AAV Vector Delivery of Tissue Optimized Transgene Cassettes (A) Schematics of AAV2/8 transgene designs. Straight lines represent DNA sequence of no known function. (B) AAV2/8-HLP-An53-LCO and AAV2/8-HLP-An53-HCO were delivered intravenously to hemophilia A mice at a dose of 1 × 10 11 vg/kg (n = 4 per group). (C) AAV2/8 HCB-HSQ-LCO, AAV2/8-HCB-ET3-LCO, and AAV2/8-HLP-V3co were delivered intravenously to hemophilia A mice at a dose of 1 × 10 11 vg/kg (n = 3–4 per group). (D) A long-term, dose-finding experiment was performed with varying does of AAV2/8-HCB-MVM-ET3-LCO (n = 3 for all other doses). Data are presented as the mean ± sample SD.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Target-Cell-Directed Bioengineering Approaches for Gene Therapy of Hemophilia A

    doi: 10.1016/j.omtm.2018.01.004

    Figure Lengend Snippet: AAV Vector Delivery of Tissue Optimized Transgene Cassettes (A) Schematics of AAV2/8 transgene designs. Straight lines represent DNA sequence of no known function. (B) AAV2/8-HLP-An53-LCO and AAV2/8-HLP-An53-HCO were delivered intravenously to hemophilia A mice at a dose of 1 × 10 11 vg/kg (n = 4 per group). (C) AAV2/8 HCB-HSQ-LCO, AAV2/8-HCB-ET3-LCO, and AAV2/8-HLP-V3co were delivered intravenously to hemophilia A mice at a dose of 1 × 10 11 vg/kg (n = 3–4 per group). (D) A long-term, dose-finding experiment was performed with varying does of AAV2/8-HCB-MVM-ET3-LCO (n = 3 for all other doses). Data are presented as the mean ± sample SD.

    Article Snippet: The synthetic AAV2 expression cassette was constructed by Genscript Biotech and inserted into the pUC57 backbone.

    Techniques: Plasmid Preparation, Sequencing, Mouse Assay

    BE-FLARE facilitates reciprocal enrichment of co-edited cells. a Enrichment for BFP editing by selection for EGFR T790 M co-editing. PC9 cells stably expressing the BE-FLARE reporter were co-transfected with a construct expressing BE3 and gRNAs targeting EGFR T790 and BFP. Cells were treated with 100 nM gefitinib 72 h after transfection to select for EGFR T790 M mutants. GFP-positive cells were quantified by flow cytometry 4 days after addition of gefitinib. b Quantification of three independent experiments from ( a ), with each data point shown and mean represented as a bar. Unpaired Student’s t test; *** P

    Journal: BMC Biology

    Article Title: BE-FLARE: a fluorescent reporter of base editing activity reveals editing characteristics of APOBEC3A and APOBEC3B

    doi: 10.1186/s12915-018-0617-1

    Figure Lengend Snippet: BE-FLARE facilitates reciprocal enrichment of co-edited cells. a Enrichment for BFP editing by selection for EGFR T790 M co-editing. PC9 cells stably expressing the BE-FLARE reporter were co-transfected with a construct expressing BE3 and gRNAs targeting EGFR T790 and BFP. Cells were treated with 100 nM gefitinib 72 h after transfection to select for EGFR T790 M mutants. GFP-positive cells were quantified by flow cytometry 4 days after addition of gefitinib. b Quantification of three independent experiments from ( a ), with each data point shown and mean represented as a bar. Unpaired Student’s t test; *** P

    Article Snippet: The BE3 expression cassette was synthesised (ThermoFisher) and cloned into pcDNA3.1(+).

    Techniques: Selection, Stable Transfection, Expressing, Transfection, Construct, Flow Cytometry, Cytometry

    Co-enrichment of editing events using BE-FLARE is superior to selecting transfected cells with BE3-T2A-RFP. a Schematic diagram of the enrichment strategy for genomic co-editing events using the BE-FLARE reporter. b ]. Data are expressed as the mean ± SD of two independent experiments. c Schematic of the methodology used to monitor base editing efficiency and BE3 expression using a BE3-T2A-TurboRFP construct (BE3-RFP). d

    Journal: BMC Biology

    Article Title: BE-FLARE: a fluorescent reporter of base editing activity reveals editing characteristics of APOBEC3A and APOBEC3B

    doi: 10.1186/s12915-018-0617-1

    Figure Lengend Snippet: Co-enrichment of editing events using BE-FLARE is superior to selecting transfected cells with BE3-T2A-RFP. a Schematic diagram of the enrichment strategy for genomic co-editing events using the BE-FLARE reporter. b ]. Data are expressed as the mean ± SD of two independent experiments. c Schematic of the methodology used to monitor base editing efficiency and BE3 expression using a BE3-T2A-TurboRFP construct (BE3-RFP). d

    Article Snippet: The BE3 expression cassette was synthesised (ThermoFisher) and cloned into pcDNA3.1(+).

    Techniques: Transfection, Expressing, Construct

    A fluorescent reporter detects base editing activity. a Diagram of the BE-FLARE reporter comprised of a modified BFP (BFP) and gRNA sequence used to transition BFP to GFP through base editing (BE). Codon 66 (CAC) encoding histidine is targeted and converted to tyrosine (codons TAT or TAC), resulting in GFP expression. Codon conversion to CAT is synonymous for His, thus the protein remains as BFP. b BFP to GFP conversion in HEK293 and PC9 cells. Cells were co-transfected with the BE-FLARE and a plasmid expressing BE3 and either a non-targeting guide (NT-BE) or a BFP targeting guide (BFP-BE). BFP and GFP positive cells were quantified by flow cytometry 72 h after transfection. Data are representative of three independent experiments. c PC9 cells from the experiment described in ( b

    Journal: BMC Biology

    Article Title: BE-FLARE: a fluorescent reporter of base editing activity reveals editing characteristics of APOBEC3A and APOBEC3B

    doi: 10.1186/s12915-018-0617-1

    Figure Lengend Snippet: A fluorescent reporter detects base editing activity. a Diagram of the BE-FLARE reporter comprised of a modified BFP (BFP) and gRNA sequence used to transition BFP to GFP through base editing (BE). Codon 66 (CAC) encoding histidine is targeted and converted to tyrosine (codons TAT or TAC), resulting in GFP expression. Codon conversion to CAT is synonymous for His, thus the protein remains as BFP. b BFP to GFP conversion in HEK293 and PC9 cells. Cells were co-transfected with the BE-FLARE and a plasmid expressing BE3 and either a non-targeting guide (NT-BE) or a BFP targeting guide (BFP-BE). BFP and GFP positive cells were quantified by flow cytometry 72 h after transfection. Data are representative of three independent experiments. c PC9 cells from the experiment described in ( b

    Article Snippet: The BE3 expression cassette was synthesised (ThermoFisher) and cloned into pcDNA3.1(+).

    Techniques: Activity Assay, Modification, Sequencing, Expressing, Transfection, Plasmid Preparation, Flow Cytometry, Cytometry

    Activation of the mouse Oct4 and Nanog loci by TALE-As and dCas9-As/gRNAs. ( A ) Schematic diagram of the dCas9-As evaluated in this study. In all cases, blue florescent protein (BFP) was used to track the expression of dCas9-As. gRNA expression is controlled by U6 promoter, and EF1a-mCherry is used to detect the integration of the vector into the genome. A blasticidin resistance and the reverse tetracycline transactivator (rtTA) cassettes were also linked to the mCherry cassette by T2A peptides. PB-5TR and PB-3TR are the two terminal repeat sequences of the piggyBac (PB) transposon. ( B ) Schematic diagram of the TALE and dCas9/gRNA targeting sites at the mouse Oct4 and Nanog enhancers. Red arrows indicate the gRNA targeting sites and the blue arrows mark the TALE sites. ( C ) Activation of the Oct4 distal enhancer luciferase reporter by the TALE-As and dCas9-As/gRNAs. ( D ) qRT-PCR analysis of the Oct4 mRNA levels in MEFs expressing the TALE-As or dCas9-As/gRNAs. ( E ) Activation of the Nanog enhancer luciferase reporter by the TALE-As and dCas9-As/gRNAs. ( F ) qRT-PCR analysis of the Nanog mRNA levels in EpiSCs expressing the TALE-As or dCas9-As/gRNAs. All gene expression values were normalized to Gapdh . Results were representative of three independent experiments and were presented as ±SD, n = 3. * P

    Journal: Nucleic Acids Research

    Article Title: Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers

    doi: 10.1093/nar/gku836

    Figure Lengend Snippet: Activation of the mouse Oct4 and Nanog loci by TALE-As and dCas9-As/gRNAs. ( A ) Schematic diagram of the dCas9-As evaluated in this study. In all cases, blue florescent protein (BFP) was used to track the expression of dCas9-As. gRNA expression is controlled by U6 promoter, and EF1a-mCherry is used to detect the integration of the vector into the genome. A blasticidin resistance and the reverse tetracycline transactivator (rtTA) cassettes were also linked to the mCherry cassette by T2A peptides. PB-5TR and PB-3TR are the two terminal repeat sequences of the piggyBac (PB) transposon. ( B ) Schematic diagram of the TALE and dCas9/gRNA targeting sites at the mouse Oct4 and Nanog enhancers. Red arrows indicate the gRNA targeting sites and the blue arrows mark the TALE sites. ( C ) Activation of the Oct4 distal enhancer luciferase reporter by the TALE-As and dCas9-As/gRNAs. ( D ) qRT-PCR analysis of the Oct4 mRNA levels in MEFs expressing the TALE-As or dCas9-As/gRNAs. ( E ) Activation of the Nanog enhancer luciferase reporter by the TALE-As and dCas9-As/gRNAs. ( F ) qRT-PCR analysis of the Nanog mRNA levels in EpiSCs expressing the TALE-As or dCas9-As/gRNAs. All gene expression values were normalized to Gapdh . Results were representative of three independent experiments and were presented as ±SD, n = 3. * P

    Article Snippet: To generate the PB-U6-gRNA–EFα-mCherry-2A-rtTA-2A-BSD vector, the U6 promoter driven gRNA expression cassette (Addgene 44248) was first cloned into the PB-LTR vector.

    Techniques: Activation Assay, Expressing, Plasmid Preparation, Luciferase, Quantitative RT-PCR

    Repression of the Oct4 and Nanog loci by TALE-Rs and dCas9-Rs/gRNAs. ( A ) Schematic diagram of the dCas9-R designs evaluated in this study. In all cases, blue florescent protein (BFP) was used to track the expression of dCas9-R. The gRNA vector used was the same as described in Figure 1 . ( B ) Repression of the endogenous Oct4 locus in Oct4-GFP ES cells indicated by the reduction of GFP intensity in flow cytometric analysis on day 0 and day 3 of expression of the TALE-Rs or dCas9-Rs/gRNAs targeting at the Sites 1–4 of the Oct4 distal enhancer (Gated mCherry + for TALEs and mCherry + /BFP + for dCas9-As/gRNAs). ( C ) Comparison of Oct4 expression levels in Oct4-GFP ES cells expressing the TALE-Rs or dCas9-Rs targeting at the Site 1 or Site 3 of the Oct4 distal enhancer by qRT-PCR. ( D ) The repressive effect of TALE-R and dCas9-Rs/gRNAs targeting at the Site 3 of the Oct4 distal enhancer on MEF reprogramming. MEFs were reprogrammed with Dox inducible CKS and Lrh1 (CKSL) factors together with the TALE-R or dCas9-R/gRNAs as in (C). ‘CKSL+’ indicates that all transfections have CKSL. ‘−’ is the CKSL only control (no repressor). ‘CKS only’ is the reprogramming negative control. ( E ) Reprogramming using FACS-sorted MEFs (as described in 2E) to control transfection variability. Wild-type MEFs were transfected and sorted on day 2 for GFP + /mCherry + in the TALE-R transfection and for GFP + /mCherry + /BFP + in dCas9-R/gRNAs (PL-R) experiments. Both TALE-Rs and dCas9-R/gRNAs (PL-R) targeted the Site 2–4 of the Oct4 distal enhancer. Sorted MEFs were re-plated (20 000 cells/ well) for reprogramming and iPSC colonies were scored by AP staining. ( F ) The repressive effect of TALE-Rs or dCas9-Rs/gRNAs targeting at the Sites 1–2 of the Nanog 5 kb enhancer in Nanog-GFP ES cells. Site 1 is located outside the enhancer region whereas Site 2 is inside. The Nanog repression was demonstrated by the increase of the GFP low/dim fraction in Nanog-GFP ES cells. ( G ) Endogenous N anog mRNA levels in Nanog-GFP ES cells expressing the TALE-Rs or dCas9-Rs targeting at the Sites 1–2. ( H ) Repression of Klf4- mediated EpiSC reprogramming to iPSCs by the TALE-R and dCas9-Rs targeting at the Site 2 of the Nanog 5 kb enhancer. ‘Klf4+’ refers to the transfections combined with Klf4 and ‘−’ is the Klf4 control (no effector). Results were representative of three independent experiments and were presented as ±SD, n = 3.

    Journal: Nucleic Acids Research

    Article Title: Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers

    doi: 10.1093/nar/gku836

    Figure Lengend Snippet: Repression of the Oct4 and Nanog loci by TALE-Rs and dCas9-Rs/gRNAs. ( A ) Schematic diagram of the dCas9-R designs evaluated in this study. In all cases, blue florescent protein (BFP) was used to track the expression of dCas9-R. The gRNA vector used was the same as described in Figure 1 . ( B ) Repression of the endogenous Oct4 locus in Oct4-GFP ES cells indicated by the reduction of GFP intensity in flow cytometric analysis on day 0 and day 3 of expression of the TALE-Rs or dCas9-Rs/gRNAs targeting at the Sites 1–4 of the Oct4 distal enhancer (Gated mCherry + for TALEs and mCherry + /BFP + for dCas9-As/gRNAs). ( C ) Comparison of Oct4 expression levels in Oct4-GFP ES cells expressing the TALE-Rs or dCas9-Rs targeting at the Site 1 or Site 3 of the Oct4 distal enhancer by qRT-PCR. ( D ) The repressive effect of TALE-R and dCas9-Rs/gRNAs targeting at the Site 3 of the Oct4 distal enhancer on MEF reprogramming. MEFs were reprogrammed with Dox inducible CKS and Lrh1 (CKSL) factors together with the TALE-R or dCas9-R/gRNAs as in (C). ‘CKSL+’ indicates that all transfections have CKSL. ‘−’ is the CKSL only control (no repressor). ‘CKS only’ is the reprogramming negative control. ( E ) Reprogramming using FACS-sorted MEFs (as described in 2E) to control transfection variability. Wild-type MEFs were transfected and sorted on day 2 for GFP + /mCherry + in the TALE-R transfection and for GFP + /mCherry + /BFP + in dCas9-R/gRNAs (PL-R) experiments. Both TALE-Rs and dCas9-R/gRNAs (PL-R) targeted the Site 2–4 of the Oct4 distal enhancer. Sorted MEFs were re-plated (20 000 cells/ well) for reprogramming and iPSC colonies were scored by AP staining. ( F ) The repressive effect of TALE-Rs or dCas9-Rs/gRNAs targeting at the Sites 1–2 of the Nanog 5 kb enhancer in Nanog-GFP ES cells. Site 1 is located outside the enhancer region whereas Site 2 is inside. The Nanog repression was demonstrated by the increase of the GFP low/dim fraction in Nanog-GFP ES cells. ( G ) Endogenous N anog mRNA levels in Nanog-GFP ES cells expressing the TALE-Rs or dCas9-Rs targeting at the Sites 1–2. ( H ) Repression of Klf4- mediated EpiSC reprogramming to iPSCs by the TALE-R and dCas9-Rs targeting at the Site 2 of the Nanog 5 kb enhancer. ‘Klf4+’ refers to the transfections combined with Klf4 and ‘−’ is the Klf4 control (no effector). Results were representative of three independent experiments and were presented as ±SD, n = 3.

    Article Snippet: To generate the PB-U6-gRNA–EFα-mCherry-2A-rtTA-2A-BSD vector, the U6 promoter driven gRNA expression cassette (Addgene 44248) was first cloned into the PB-LTR vector.

    Techniques: Expressing, Plasmid Preparation, Flow Cytometry, Quantitative RT-PCR, Transfection, Negative Control, FACS, Staining