Journal:

Article Title: A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen

doi: 10.1073/pnas.1131930100

Figure Lengend Snippet: HeLa cells internalize LFn-GFP in the absence of PA, but do not internalize GFP. HeLa cells were incubated with Texas red-conjugated transferrin and GFP (a and c) or LFn-GFP (b and d) for 1 hr (a and b) or 2 hr (c and d) and imaged by confocal microscopy. (Right) Overlay of red and green staining. (Center) Uptake of transferrin in red. (Left) Internalized LFn-GFP or GFP in green. Similar results were found for COS cells (data not shown).

Article Snippet: HeLa cells (American Type Culture Collection), grown on collagen-treated chamber slides (BD Science) to reach ≈80% confluence, were incubated with 40 μg/ml purified GFP or LFn-GFP at 37°C for 1 or 2 h. Some incubations were performed in the presence of 100 μg/ml Texas red-conjugated transferrin (Molecular Probes) as a marker for the endocytic pathway or with 10 μg/ml PA. For the transferrin experiments, cells were washed four times with cold DMEM and then fixed for 15 min in 4% paraformaldehyde in cold PBS.

Techniques: Incubation, Confocal Microscopy, Staining

Journal:

Article Title: A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen

doi: 10.1073/pnas.1131930100

Figure Lengend Snippet: Internalized LFn-GFP does not completely localize with the endocytic or secretory pathways. HeLa cells incubated for 1 hr with LFn-GFP in the absence of PA were stained with markers for early endosomes (EEA-1; a), late endosomes (Lamp-1; b), lysosomes (Lamp-2; c), and the Golgi apparatus (Ab-1; d) and visualized by confocal microscopy. (Right) Overlay of red and green staining. (Center) Red organelle antibody staining. (Left) Green fluorescence of LFn-GFP.

Article Snippet: HeLa cells (American Type Culture Collection), grown on collagen-treated chamber slides (BD Science) to reach ≈80% confluence, were incubated with 40 μg/ml purified GFP or LFn-GFP at 37°C for 1 or 2 h. Some incubations were performed in the presence of 100 μg/ml Texas red-conjugated transferrin (Molecular Probes) as a marker for the endocytic pathway or with 10 μg/ml PA. For the transferrin experiments, cells were washed four times with cold DMEM and then fixed for 15 min in 4% paraformaldehyde in cold PBS.

Techniques: Incubation, Staining, Confocal Microscopy, Fluorescence

Journal:

Article Title: A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen

doi: 10.1073/pnas.1131930100

Figure Lengend Snippet: Colocalization of LFn-GFP with the proteosome 20s subunit. HeLa cells were stained with the mixture of an antibody to the α-subunit and the other to the β-subunit of the 20s proteosome 2 hr after incubation with LFn-GFP. Similar results were also found after 1 hr incubation (see Fig. 1) and with a single antibody to the α-subunit or β-subunit of the proteosome, respectively (data not shown). (Top) Green fluorescence of LFn-GFP. (Middle) Red fluorescence of the proteosome antibody. (Bottom) Overlay of both channels.

Article Snippet: HeLa cells (American Type Culture Collection), grown on collagen-treated chamber slides (BD Science) to reach ≈80% confluence, were incubated with 40 μg/ml purified GFP or LFn-GFP at 37°C for 1 or 2 h. Some incubations were performed in the presence of 100 μg/ml Texas red-conjugated transferrin (Molecular Probes) as a marker for the endocytic pathway or with 10 μg/ml PA. For the transferrin experiments, cells were washed four times with cold DMEM and then fixed for 15 min in 4% paraformaldehyde in cold PBS.

Techniques: Staining, Incubation, Fluorescence

Journal:

Article Title: A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen

doi: 10.1073/pnas.1131930100

Figure Lengend Snippet: Internalization and colocalization of LFn-GFP with the proteosome 20s subunit is more efficient in the absence of PA. HeLa cells were stained for the proteosome 20s α-subunit 1 hr (a and b)or2hr(c and d) after incubation with LFn-GFP in the absence (a and c) or presence (b and d) of PA. (Right) Overlay. (Center) Red fluorescent staining for the proteosome. (Left) Green fluorescence of LFn-GFP.

Article Snippet: HeLa cells (American Type Culture Collection), grown on collagen-treated chamber slides (BD Science) to reach ≈80% confluence, were incubated with 40 μg/ml purified GFP or LFn-GFP at 37°C for 1 or 2 h. Some incubations were performed in the presence of 100 μg/ml Texas red-conjugated transferrin (Molecular Probes) as a marker for the endocytic pathway or with 10 μg/ml PA. For the transferrin experiments, cells were washed four times with cold DMEM and then fixed for 15 min in 4% paraformaldehyde in cold PBS.

Techniques: Staining, Incubation, Fluorescence

Journal: Nature Communications

Article Title: Characterizing a thermostable Cas9 for bacterial genome editing and silencing

doi: 10.1038/s41467-017-01591-4

Figure Lengend Snippet: The Geobacillus thermodenitrificans T12 type-IIC CRISPR-Cas locus encoding ThermoCas9. a Schematic representation of the genomic locus encoding ThermoCas9. The domain architecture of ThermoCas9 based on sequence comparison, with predicted active sites residues highlighted in magenta. A homology model of ThermoCas9 generated using Phyre 2 is shown, with different colors for the domains. b Phylogenetic tree of Cas9 orthologues highly identical to ThermoCas9. Evolutionary analysis was conducted in MEGA7 . c SDS-PAGE of ThermoCas9 after purification by metal-affinity chromatography and gel filtration. The migration of the obtained single band is consistent with the theoretical molecular weight of 126 kD of the apo-ThermoCas9

Article Snippet: The gene sequence was inserted into plasmid pML-1B (obtained from the UC Berkeley MacroLab, Addgene #29653) by ligation-independent cloning using oligonucleotides (Supplementary 2) to generate a protein expression construct encoding the ThermoCas9 polypeptide sequence (residues 1–1082) fused with an N-terminal tag comprising a hexahistidine sequence and a Tobacco Etch Virus (TEV) protease cleavage site.

Techniques: CRISPR, Sequencing, Comparison, Generated, SDS Page, Purification, Affinity Chromatography, Filtration, Migration, Molecular Weight

Journal: Nature Communications

Article Title: Characterizing a thermostable Cas9 for bacterial genome editing and silencing

doi: 10.1038/s41467-017-01591-4

Figure Lengend Snippet: ThermoCas9 PAM analysis. a Schematic illustrating the in vitro cleavage assay for discovering the position and identity (5′-NNNNNNN-3′) of the protospacer adjacent motif (PAM). Magenta triangles indicate the cleavage position. b Sequence logo of the consensus 7 nt long PAM of ThermoCas9, obtained by comparative analysis of the ThermoCas9-based cleavage of target libraries. Letter height at each position is measured by information content. c Extension of the PAM identity to the eighth position by in vitro cleavage assay. Four linearized plasmid targets, each containing a distinct 5′-CCCCCCAN-3′ PAM, were incubated with ThermoCas9 and sgRNA at 55 °C for 1 h, then analyzed by agarose gel electrophoresis. Supplementary Fig. shows the uncropped gel image. d In vitro cleavage assays for DNA targets with different PAMs at 30 and 55 °C. Sixteen linearized plasmid targets, each containing one distinct 5′-CCCCCNNA-3′ PAM, were incubated with ThermoCas9 and sgRNA, then analyzed for cleavage efficiency by agarose gel electrophoresis. See also Supplementary Fig. . Supplementary Fig. shows the uncropped gel images

Article Snippet: The gene sequence was inserted into plasmid pML-1B (obtained from the UC Berkeley MacroLab, Addgene #29653) by ligation-independent cloning using oligonucleotides (Supplementary 2) to generate a protein expression construct encoding the ThermoCas9 polypeptide sequence (residues 1–1082) fused with an N-terminal tag comprising a hexahistidine sequence and a Tobacco Etch Virus (TEV) protease cleavage site.

Techniques: In Vitro, Cleavage Assay, Sequencing, Plasmid Preparation, Incubation, Agarose Gel Electrophoresis

Journal: Nature Communications

Article Title: Characterizing a thermostable Cas9 for bacterial genome editing and silencing

doi: 10.1038/s41467-017-01591-4

Figure Lengend Snippet: ThermoCas9 temperature range and effect of sgRNA-binding. a Schematic representation of the sgRNA and a matching target DNA. The target DNA, the PAM and the crRNA are shown in gray, blue and turquoise, respectively. The site where the crRNA is linked with the tracrRNA is shown in purple. The dark blue and light blue boxes indicate the predicted three and two loops of the tracrRNA, respectively. The 41-nt truncation of the repeat-anti-repeat region and the three loops of the sgRNA are indicated by the magenta dotted line and magenta triangles, respectively. b The importance of the predicted three stem-loops of the tracrRNA scaffold was tested by transcribing truncated variants of the sgRNA and evaluating their ability to guide ThermoCas9 to cleave target DNA at various temperatures. Average values of three replicates are shown, with error bars representing S.D. The blots of one of the replicates are shown in Supplementary Fig. . c The importance of the length of the spacer was tested by transcribing truncated variants of the initial spacer in the sgRNA and evaluating their ability to guide ThermoCas9 to cleave target DNA at 55 °C. Average values of three replicates are shown, with error bars representing S.D.. The blots of one of the replicates are shown in Supplementary Fig. . d To identify the maximum temperature, endonuclease activity of ThermoCas9:sgRNA RNP complex was assayed after incubation at 60, 65, and 70 °C for 5 or 10 min. The pre-heated DNA substrate was added and the reaction was incubated for 1 h at the corresponding temperature. e Comparison of active temperature range of ThermoCas9 and SpCas9 by activity assays conducted after 5 min of incubation at the indicated temperature. The pre-heated DNA substrate was added and the reaction was incubated for 1 h at the same temperature

Article Snippet: The gene sequence was inserted into plasmid pML-1B (obtained from the UC Berkeley MacroLab, Addgene #29653) by ligation-independent cloning using oligonucleotides (Supplementary 2) to generate a protein expression construct encoding the ThermoCas9 polypeptide sequence (residues 1–1082) fused with an N-terminal tag comprising a hexahistidine sequence and a Tobacco Etch Virus (TEV) protease cleavage site.

Techniques: Binding Assay, Activity Assay, Incubation, Comparison

Journal: Nature Communications

Article Title: Characterizing a thermostable Cas9 for bacterial genome editing and silencing

doi: 10.1038/s41467-017-01591-4

Figure Lengend Snippet: Protospacer targeting Specificity of ThermoCas9. a Scheme of the generated mismatch protospacers library, employed for evaluating the ThermoCas9:sgRNA targeting specificity in vitro. The mismatch spacer-protospacer positions are shown in red, the PAM in light blue with the fifth to eighth positions underlined. b Graphical representation of the ThermoCas9:sgRNA cleavage efficiency over linear or plasmid targets with different mismatches at 37 °C. The percentage of cleavage was calculated based on integrated band intensities after gel electrophoresis. Average values from three biological replicates are shown, with error bars representing S.D. The blots of one of the replicates are shown in Supplementary Fig. . c Graphical representation of the ThermoCas9:sgRNA cleavage efficiency over linear or plasmid targets with different mismatches at 55 °C. The percentage of cleavage was calculated based on integrated band intensities after gel electrophoresis. Average values from three biological replicates are shown, with error bars representing S.D. The blots of one of the replicates are shown in Supplementary Fig.

Article Snippet: The gene sequence was inserted into plasmid pML-1B (obtained from the UC Berkeley MacroLab, Addgene #29653) by ligation-independent cloning using oligonucleotides (Supplementary 2) to generate a protein expression construct encoding the ThermoCas9 polypeptide sequence (residues 1–1082) fused with an N-terminal tag comprising a hexahistidine sequence and a Tobacco Etch Virus (TEV) protease cleavage site.

Techniques: Generated, In Vitro, Plasmid Preparation, Nucleic Acid Electrophoresis

Journal: Nature Communications

Article Title: Characterizing a thermostable Cas9 for bacterial genome editing and silencing

doi: 10.1038/s41467-017-01591-4

Figure Lengend Snippet: ThermoCas9-based genome engineering in prokaryotes. a Schematic overview of the basic pThermoCas9_Δgene-of-interest (goi) construct. The thermocas9 gene was introduced either to the pNW33n ( B. smithii ) or to the pEMG ( P. putida ) vector. Homologous recombination flanks were introduced upstream thermocas9 and encompassed the 1 kb ( B. smithii ) or 0.5 kb ( P. putida ) upstream and 1 kb or 0.5 kb downstream region of the gene of interest (goi) in the targeted genome. A sgRNA-expressing module was introduced downstream the thermocas9 gene. As the origin of replication (ori), replication protein (rep), antibiotic resistance marker (AB) and possible accesory elements (AE) are backbone specific, they are represented with dotted outline. b Agarose gel electrophoresis showing the resulting products from genome-specific PCR on ten colonies from the ThermoCas9-based pyrF deletion process from the genome of B. smithii ET 138. All ten colonies contained the Δ pyrF genotype and one colony was a clean Δ pyrF mutant, lacking the wild-type product. Supplementary Fig. shows the uncropped gel image. c Schematic overview of the basic pThermoCas9i_goi construct. Aiming for the expression of a catalytically inactive ThermoCas9 (ThermodCas9: D8A, H582A mutant), the corresponding mutations were introduced to create the thermodcas9 gene. The thermodcas9 gene was introduced to the pNW33n vector. A sgRNA-expressing module was introduced downstream the thermodcas9 . d Graphical representation of the production, growth and RT-qPCR results from the ldhL silencing experiment using ThermodCas9. The graphs represent the lactate production, optical density at 600 nm and percentage of ldhL transcription in the repressed cultures compared to the control cultures. Average values from three biological replicates are shown, with error bars representing S.D

Article Snippet: The gene sequence was inserted into plasmid pML-1B (obtained from the UC Berkeley MacroLab, Addgene #29653) by ligation-independent cloning using oligonucleotides (Supplementary 2) to generate a protein expression construct encoding the ThermoCas9 polypeptide sequence (residues 1–1082) fused with an N-terminal tag comprising a hexahistidine sequence and a Tobacco Etch Virus (TEV) protease cleavage site.

Techniques: Construct, Plasmid Preparation, Homologous Recombination, Expressing, Marker, Agarose Gel Electrophoresis, Mutagenesis, Quantitative RT-PCR, Control