Journal: Nature communications

Article Title: Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells.

doi: 10.1038/ncomms7413

Figure Lengend Snippet: Figure 1 | CRISPR/Cas9 directed disruption of lentivirus infection. (a) Model of CRISPR/Cas9 directed intracellular defense against lentiviral infection. hCas9 and its gRNA can be synthesized from either transfected plasmids (module A) or knockin expression cassettes (module B). After synthesis, hCas9 and gRNA assemble and work as an antiviral module in different steps. Once penetrating into the host cells, virally encoded reverse transcriptase uses the lentiviral RNA genome as a template for the synthesis of viral cDNA. Viral integrase binds and processes the LTR and assists in the insertion of the viral cDNA into the host genome. During these processes, the viral genome is unprotected by its envelope and capsid, rendering the viral cDNA vulnerable to cleavage by DNA endonucleases. In our study, the CRISPR/Cas9 system was adapted for human cells as an antiviral module, which bound and disrupted the viral genome of both pre-integration viral DNA (Step 1) and integrated provirus (Step 2). (b) Seven gRNAs were designed and used with hCas9 to target corresponding regions in the EGFP reporter lentivirus, including gEGFP-T1 to -T4 and gLTR-T1 to -T3. (c) FACS analysis of HEK293 cells that were pretreated with hCas9-mCh and gRNAs for 20B24 h, and then challenged by EGFP reporter lentiviruses for 4 days. gEmpty is an empty gRNA vector and gMock (as the gS35 in Supplementary Fig. 1) is a non-targeting mock gRNA. (d–g) The CRISPR/Cas9 system can direct targeted disruption of the non- integrative lentivirus. (d) Fluorescence microscopy images of HEK293 cells pretreated with CRISPR/Cas9 followed by non-integrative lentivirus infection for 3 days. Insets show bright field images. Scale bar, 500 mm. (e) EGFP expressions of invading non-integrated lentivirus were measured by flow- cytometric analysis. Overlay histogram depicts the gEmpty in the first panel and the dashed blue line indicates the non-treatment cells. (f) The bar graph represents the average of MFI (mean fluorescence intensity) from triplicates of FACS analysis. Statistical analysis determined using unpaired t-test (***Po0.001; **Po0.01) (g) Time-course analysis of EGFP expression from integrative and non-integrative lentiviruses without any treatment with CRISPR/Cas9. The results showed that the integrative lenti-EGFP expressed constitutively from 7 days after infection, whereas the expression of non- integrative lenti-EGFP gradually reduced over time.

Article Snippet: For lentiviral vector production, EGFP expression plasmid (pEGIP) was purchased from Addgene (29777)20. pEGIP plasmid was packaged and purified as a lentiviral vector according to published papers and the vesicular stomatitis virus Env glycoprotein (VSV-G) is typically used41.

Techniques: CRISPR, Disruption, Infection, Synthesized, Transfection, Knock-In, Expressing, Reverse Transcription, Plasmid Preparation, Fluorescence, Microscopy

Journal: Nature communications

Article Title: Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells.

doi: 10.1038/ncomms7413

Figure Lengend Snippet: Figure 2 | CRISPR/Cas9 directed disruption of integrated lentivirus. CRISPR/Cas9-mediated disruption of integrated lenti-proviral DNA in infected HEK293 cells. (a) Top: confocal images of cells with high copy numbers of EGFP proviral DNA that were co-transfected with hCas9 and either gEGFP-T1 (right panels) or mock gRNA (middle panels). Left panels show untreated cells. Images were taken at 14 days after transfection. Bottom: confocal images with DAPI staining (blue). Scale bars, 50 mm. (b) Determination of provirus copy numbers in three different lentiviral integrated HEK293 cell lines. (c) PCR analysis of proviral DNA. The amount of full-length provirus (arrow) was reduced in the GFP-negative cells generated by CRISPR/Cas9-mediated lentiviral disruption. (d) DNA sequencing analysis of the CRISPR/Cas9 target sites in the EGFP proviruses. Two examples shown here were treated with hCas9 and either gEGFP-T1 (top) or gLTR-T2 (bottom). Sequences identified multiple times were marked accordingly on the right. (e) Proviral copy number quantitation by qPCR. Statistical analysis determined using unpaired t-test (***Po0.001; **Po0.01; *Po0.1). (f) FACS analysis of proviral EGFP expression at different days after transfection of hCas9 and gRNAs into cells with either high or low provirus copy numbers as determined in b. (g) FACS analysis of EGFP expression at different days after treatment of the integrated lenti-EGFP HEK293 cell lines with hCas9-mCh and the corresponding gRNA. CRISPR/ Cas-mediated disruption of integrated lentiviral DNA can be augmented by either multiplexed site-directed targeting or combinatorial treatments. gEGFP- T1-gEGFP-T3 and gEGFP-T1-gLTR-T2 represent multiplexed targeting. In addition, to examine the effects of multiple treatments, we introduced secondary transfection of hCas9 together with the indicated gRNAs into provirus Lenti-EGFP cells that were pre-treated with hCas9-gEGFP-T1 for 18 days.

Article Snippet: For lentiviral vector production, EGFP expression plasmid (pEGIP) was purchased from Addgene (29777)20. pEGIP plasmid was packaged and purified as a lentiviral vector according to published papers and the vesicular stomatitis virus Env glycoprotein (VSV-G) is typically used41.

Techniques: CRISPR, Disruption, Infection, Transfection, Staining, Generated, DNA Sequencing, Quantitation Assay, Expressing

Journal: Nature communications

Article Title: Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells.

doi: 10.1038/ncomms7413

Figure Lengend Snippet: Figure 3 | CRISPR/Cas9-directed disruption of HIV-1 virus. (a) HIV virus constructs used to generate the pseudotyped (HIV-1NL4-3-DE-GFP) and wild-type reporter virus (HIV-1NL43-GFP) for infection in this study. (b) FACS analysis of the average of MFI (mean fluorescence intensity) of EGFP in HIV-1NL4-3-DE-GFP infected HEK293 cells transfected with hCas9 together with a variety of gRNAs against the HIV genome. See Supplementary Table 1 for details about the gRNAs. (c) Comparison of antiviral disruption with single or double gRNAs performed in 293T.CD4.CCR5 cells infected with HIV-1NL4-3-DE-GFP. (d) Cell viability analysis of cells containing CRISPR/Cas9 following HIV-1 infection. Bright-field and fluorescent images show that gEGFP-T1-gLTR-T2 HEK293 cells are resistant to HIV-1NL4-3-DE-GFP infection and have higher cell viability (bottom panels on the left and graph on the right) when compared with gMock treated cells (top panels). Scale bars, 100 mm. (e) CRISPR/Cas9-mediated antiviral activity against wild-type HIV-1 reporter virus (HIV-1NL43-GFP) was monitored by the levels of the viral p24 protein (top) and GFP reporter expression (bottom). All statistical comparisons were made by unpaired t-test (***Po0.001; **Po0.01; *Po0.1; NS, no significant) with at least three replicates.

Article Snippet: For lentiviral vector production, EGFP expression plasmid (pEGIP) was purchased from Addgene (29777)20. pEGIP plasmid was packaged and purified as a lentiviral vector according to published papers and the vesicular stomatitis virus Env glycoprotein (VSV-G) is typically used41.

Techniques: CRISPR, Disruption, Virus, Construct, Infection, Transfection, Comparison, Activity Assay, Expressing

Journal: Cell reports

Article Title: Mesothelial Cell HIF1α Expression Is Metabolically Downregulated by Metformin to Prevent Oncogenic Tumor-Stromal Crosstalk

doi: 10.1016/j.celrep.2019.11.079

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Recombinant DNA HA-HIF1α P402A/P564A-pcDNA3 Yan et al., 2007 Addgene: 338299 pH IV-ODD-EGFP-IRES-dTomato Bagnall et al., 2014 Addgene: 395015 SUCLG2 DNASU HsCD00445557 CXCR1 Sino Biological HG10856-NF CCR2 Sino Biological HG16084-NF EF1a-GFP-IRES-Puro (pEGIP) Zou et al., 2009 Addgene: 26777 Software and Algorithms GraphPad Prism 7 GraphPad Software RRID: SCR_002798 FIJI [ImageJ] National Institutes of Health RRID:SCR_002285 Aperio ScanScope / Pixel Count v9 Leica Biosystems RRID: SCR_014311 Thermo Xcalibur ThermoFisher Scientific RRID: SCR_014593 MaxQuant Max Planck Institute RRID: SCR_014485 Perseus Max Planck Institute RRID: SCR_015753 Molecular Signatures Database Broad Institute, MIT RRID: SCR_016863 MetaboAnalyst McGill University RRID: SCR_015539 Other Invasion transwell insert (8μm pore) ThermoFisher Scientific 353097 Co-culture transwell insert (0.4μm pore) Thomas Scientific 3450 Collagen type 1 [rat tail] Corning 354236 PureLink HiPure Plasmid Maxiprep Kit ThermoFisher Scientific K2100-06 IDEXX CellCheck IDEXX Bioresearch https://www.idexxbioanalytics.com/cellcheck Open in a separate window KEY RESOURCES TABLE Western Immunoblotting Cell lysates were prepared in 125μL RIPA buffer (100mM Tris/HCl [pH 7.4], 150mM NaCl, 1mM EDTA, 0.1% (w/v) sodium dodecyl sulfate, 0.5% sodium deoxycholate, 1% Triton X-100 and supplemented with phosphatase/protease inhibitors immediately prior to use).

Techniques: Ex Vivo, Isolation, Microarray, Recombinant, Mutagenesis, Enzyme-linked Immunosorbent Assay, Software, Plasmid Preparation