Journal: bioRxiv

Article Title: Beta human papillomavirus 8E6 promotes alternative end-joining

doi: 10.1101/2022.07.29.501983

Figure Lengend Snippet: (A) Schematic of Alt-EJ reporter. GFP is disrupted by a 46 nt insertion. One CAS9 is used to induce an upstream DSB (5’ End) and another CAS9 is used to induce a downstream DSB (either Imbedded or Terminal). Following CAS9 expression, a 4 nt microhomology mediated Alt-EJ event can restore GFP expression. (B) Representative images of flow cytometry results of HFK cells that are GFP positive 24 hours after transfection with Terminal Alt-EJ. The gating represents GFP positive based off mock transfected control. The x-axis shows cells distributed by forward scatter to avoid debris. (C) Percentage of HFK cells that are positive for GFP following transfection with Terminal Alt-EJ determined by flow cytometry. (D) Representative images of flow cytometry results of HFK cells that are GFP positive 24 hours after transfection with Imbedded Alt-EJ. The gating represents GFP positive based off mock transfected control. The x-axis shows cells distributed by forward scatter to avoid debris. (E) Percentage of HFK cells that are positive for GFP following transfection with Imbedded Alt-EJ determined by flow cytometry. All values are represented as mean ± standard error. The statistical significance of differences between cell lines were determined using Student’s t- test. * indicates significant difference between cell lines (p< 0.05). Twenty thousand cells were counted for each of three independent flow cytometry experiments.

Article Snippet: CAS9 (65832S, Cell Signaling).

Techniques: Expressing, Flow Cytometry, Transfection

Journal: Nature Communications

Article Title: Genome-wide CRISPR/Cas9 library screening identified PHGDH as a critical driver for Sorafenib resistance in HCC

doi: 10.1038/s41467-019-12606-7

Figure Lengend Snippet: CRISPR library screening identified PHGDH as a driver for Sorafenib resistance. a Schematic diagram illustrates the workflow of genome-wide CRISPR/Cas9 knockout library screening (CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats). Human genome-wide CRISPR/Cas9 knockout library (GeCKO v2A) containing 65,386 sgRNAs was packed into lentiviral particle and transduced into Cas9-overexpressing MHCC97L cells (MHCC97L-Cas9) at low multiplicity of infection (MOI). The sgRNA transduced cells were selected by puromycin to generate a mutant cell pool. Mutant cells were cultured in vehicle and Sorafenib for 7 days for genetic screening. Genomic DNA was extracted from the treated cells and the sgRNA fragment was amplified by PCR. Copy number of sgRNAs was determined by high-throughput sequencing and analyzed by MAGeCK v0.5.7 algorithm. b PHGDH (phosphoglycerate dehydrogenase) was identified as the most significant gene in the library screening as indicated by the red dot. The sgRNAs targeting PHGDH were consistently depleted in Sorafenib-treated cells. c Volcano plots revealed that PHGDH targeting sgRNAs were negatively selected during Sorafenib treatment, suggesting that PHGDH is an essential gene for HCC cells to survive from Sorafenib treatment. Source data are provided as a Source Data file

Article Snippet: The expression of Cas9 protein was confirmed by a Cas9-specific antibody (Cell Signaling #65832, dilution 1:1000).

Techniques: CRISPR, Library Screening, Genome Wide, Knock-Out, Infection, Mutagenesis, Cell Culture, Amplification, Next-Generation Sequencing

Journal: Nature Communications

Article Title: Genome-wide CRISPR/Cas9 library screening identified PHGDH as a critical driver for Sorafenib resistance in HCC

doi: 10.1038/s41467-019-12606-7

Figure Lengend Snippet: Depletion of PHGDH- sensitized HCC cells to Sorafenib treatment. a Knockout of PHGDH in MHCC97L cells by CRISPR/Cas9 gene editing system. b Knockout of PHGDH showed mild effect on cell proliferation in in vitro cell culture. However, knockout of PHGDH significantly suppressed MHCC97L cell proliferation in the presence of Sorafenib (black connected dots: non-target control; red connected dots: sgPHGDH#2; deep blue connected dots: sgPHGDH#3; green connected dots: sgPHGDH#4; purple connected dots: sgPHGDH#12; light blue connected dots: sgPHGDH#32). c Knockout of PHGDH induced apoptosis significantly upon Sorafenib treatment (gray bar: vehicle-treated group; red bar: Sorafenib-treated group). d Knockdown of PHGDH by lentiviral-based shRNA approach. e Knockdown of PHGDH suppressed HCC cell proliferation under Sorafenib treatment (black connected dots: non-target control; red connected dots: shPHGDH#20; deep blue connected dots: shPHGDH#32). f Knockdown of PHGDH augmented Sorafenib induced apoptosis in HCC cells (gray bar: vehicle-treated group; red bar: Sorafenib-treated group). g Knockdown of PHGDH sensitized HCC cell to Sorafenib in nude mice (gray connected dots: Vehicle-NTC, non-target control treated with vehicle; blue connected dots: Sora-NTC, non-target control treated with Sorafenib; red connected dots: Vehicle-shPHGDH, PHGDH knockdown clones treated with vehicle; purple connected dots: Sora-shPHGDH, PHGDH knockdown clones treated with Sorafenib). The error bar in panels b , c , d , f represents the standard error of mean (SEM), n = 3 biologically independent samples. The error bar in panel g represents the standard deviation (SD), n = 6 mice. Source data are provided as a Source Data file. (Student's t -test * P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: The expression of Cas9 protein was confirmed by a Cas9-specific antibody (Cell Signaling #65832, dilution 1:1000).

Techniques: Knock-Out, CRISPR, In Vitro, Cell Culture, shRNA, Clone Assay, Standard Deviation

Journal: Oncology Letters

Article Title: A tetracycline-inducible CRISPR/Cas9 system, targeting two long non-coding RNAs, suppresses the malignant behavior of bladder cancer cells

doi: 10.3892/ol.2018.9157

Figure Lengend Snippet: DNA sequencing was performed following transfection of cells with different gRNAs and Cas9. (A) The locations of the gRNA targets on PVT1 and ANRIL. Overlapping peaks were evident when the CRISPR/Cas9 system generated mutations in the PVT1 and ANRIL sequences using (B) gRNA1, (C) gRNA2, (D) gRNA3, (E) gRNA4 and (F) gRNA5. No change was observed in PVT1 targeted by gRNA2. There was no effect on the DNA sequence of ANRIL following transfection with gRNA3/Cas9. gRNA, guide RNA; CRISPR, clustered regularly interspaced short palindromic repeats; Cas9, CRISPR associated protein 9.

Article Snippet: The specific primary antibody against Cas9 (Streptococcus pyogenes) (clone no. D8Y4K) rabbit mAb (cat. no. 65832) and GAPDH (clone no. D16H11) XP ® Rabbit mAb (cat. no. 5174) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA; both dilutions, 1:1,000).

Techniques: DNA Sequencing, Transfection, CRISPR, Generated, Sequencing

Journal: Oncology Letters

Article Title: A tetracycline-inducible CRISPR/Cas9 system, targeting two long non-coding RNAs, suppresses the malignant behavior of bladder cancer cells

doi: 10.3892/ol.2018.9157

Figure Lengend Snippet: The relative expression levels of PVT1, ANRIL and Cas9 following transfection with tetracycline-inducible CRISPR/Cas9. (A) The expression levels of PVT1 and ANRIL in T24 cells following transfection with different gRNAs and tetracycline-inducible Cas9. (B) The mRNA expression levels of PVT1 and ANRIL in 5637 cells following transfection with different gRNAs and tetracycline-inducible Cas9. The expression levels of gRNA1, gRNA2, gRNA3, gRNA4 and gRNA5 were measured, compared with the gRNA empty vector group. gRNA3 and gRNA4 targeting PVT1, and gRNA2 and gRNA5 targeting ANRIL significantly inhibited PVT1 and ANRIL expression compared with the negative control (gRNA empty vector group) in T24 and 5637 cells. (C) The expression levels of PVT1 and ANRIL were significantly suppressed following addition of DOX in T24 cells, compared with the NC+DOX group cells (P<0.01). (D) The expression levels of PVT1 and ANRIL were significantly suppressed following addition of DOX in 5637 cells, compared with the NC+DOX group cells (P<0.01). Error bars represent the mean ± standard deviation. *P<0.05, **P<0.01. (E) In the absence of DOX, Cas9 was not expressed in T24 and 5637 cells. However, following DOX addition, expression of Cas9 was evident. CRISPR, clustered regularly interspaced short palindromic repeats; Cas9, CRISPR associated protein 9; gRNA, guide RNA; DOX, doxycycline.

Article Snippet: The specific primary antibody against Cas9 (Streptococcus pyogenes) (clone no. D8Y4K) rabbit mAb (cat. no. 65832) and GAPDH (clone no. D16H11) XP ® Rabbit mAb (cat. no. 5174) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA; both dilutions, 1:1,000).

Techniques: Expressing, Transfection, CRISPR, Plasmid Preparation, Negative Control, Standard Deviation

Journal: Oncology Letters

Article Title: A tetracycline-inducible CRISPR/Cas9 system, targeting two long non-coding RNAs, suppresses the malignant behavior of bladder cancer cells

doi: 10.3892/ol.2018.9157

Figure Lengend Snippet: Proliferation was inhibited and apoptotic rate was increased following transfection with tetracycline-inducible CRISPR/Cas9. In the absence of DOX, no significant differences were identified between the NC-DOX and Treatment-DOX groups in (A) T24 cells and (B) 5637 cells (P>0.05). With the addition of 1 µg/ml DOX to the medium, proliferation was significantly inhibited in the Treatment+DOX group compared with the NC+DOX group in T24 and 5637 cells (P<0.001). In the absence of DOX, no significant differences were identified in apoptotic rate between the NC and Treatment groups in (C) T24 and (D) 5637 cells (P>0.05). Apoptotic rate was significantly induced in the Treatment+DOX group in T24 and 5637 cells compared with the NC+DOX group (P<0.01). Error bars represent mean ± standard deviation. **P<0.01. CRISPR, clustered regularly interspaced short palindromic repeats; Cas9, CRISPR associated protein 9; DOX, doxycycline; NC, negative control; OD, optical density.

Article Snippet: The specific primary antibody against Cas9 (Streptococcus pyogenes) (clone no. D8Y4K) rabbit mAb (cat. no. 65832) and GAPDH (clone no. D16H11) XP ® Rabbit mAb (cat. no. 5174) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA; both dilutions, 1:1,000).

Techniques: Transfection, CRISPR, Standard Deviation, Negative Control

Journal: Oncology Letters

Article Title: A tetracycline-inducible CRISPR/Cas9 system, targeting two long non-coding RNAs, suppresses the malignant behavior of bladder cancer cells

doi: 10.3892/ol.2018.9157

Figure Lengend Snippet: Cell migration was suppressed following transfection with tetracycline-inducible CRISPR/Cas9. (A) In the absence of DOX, no differences in cell migration were identified between NC and Treatment groups in T24 cells. (B) The tetracycline-inducible CRISPR/Cas9 system had no effect on cell migration in the absence of DOX in 5637 cells. (C) In the absence of DOX, quantitative analysis revealed no statistical difference in cell migration between the NC and Treatment groups in T24 cells. With the addition of DOX to the medium, cell migration was significantly inhibited in the Treatment+DOX group compared with the NC+DOX group in T24 cells (P>0.05). (D) Significant suppression was observed in the Treatment+DOX group compared with the NC+DOX group in 5637 cells. Error bars represent mean ± standard deviation. **P<0.01. CRISPR, clustered regularly interspaced short palindromic repeats; Cas9, CRISPR associated protein 9; DOX, doxycycline; NC, negative control; T, time.

Article Snippet: The specific primary antibody against Cas9 (Streptococcus pyogenes) (clone no. D8Y4K) rabbit mAb (cat. no. 65832) and GAPDH (clone no. D16H11) XP ® Rabbit mAb (cat. no. 5174) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA; both dilutions, 1:1,000).

Techniques: Migration, Transfection, CRISPR, Standard Deviation, Negative Control