Journal: International Journal of Molecular Sciences

Article Title: Reversible Regulation of Polyubiquitin Gene UBC via Modified Inducible CRISPR/Cas9 System

doi: 10.3390/ijms20133168

Figure Lengend Snippet: Evaluation of UBC upregulation using the idCas9–VP64 system with sgRNAs targeting the UBC intron. ( A ) HEK293T cells were transiently transfected with idCas9–VP64 and scrambled sgRNA and then treated with 5 μg/mL Dox for up to 2 days. dCas9 mRNA levels were determined by qRT-PCR ( n = 3 each), normalized to GAPDH , and expressed as the fold change relative to the control (0 days of Dox treatment). ( B ) HEK293T cells were transiently transfected with idCas9–VP64, and scrambed sgRNA (indicated as -) or sg UBC #1~#4, alone or in combination, and then treated with 5 μg/mL Dox for 2 days. dCas9–VP64 was detected by immunoblot analysis using an anti-Cas9 antibody. α-Tubulin was used as a loading control. ( C ) Expression of scrambled sgRNA (sgScramble) or sg UBC #1~#4 was determined by conventional RT-PCR. GAPDH was used as an internal control. ( D , E ) Transient transfection of HEK293T cells and Dox treatment were performed as described in ( B ). In ( E ), HEK293T cells were treated with 10 μg/mL Dox for 2 days. UBC mRNA levels were determined by qRT-PCR ( n = 3 each), normalized to GAPDH , and expressed as the fold change relative to the control (-Dox, scrambled sgRNA). Representative immunoblots and RT-PCR results are shown from three independent experiments ( B , C ). Data are presented as means ± SEM from the indicated number of samples ( A , D , E ). ** p < 0.01; *** p < 0.001 vs. control (0 days of Dox treatment) or between two groups as indicated by horizontal bars. NS: Not significant.

Article Snippet: Using the Dox-inducible Cas9 expression vector (pCW-Cas9-Puro, #50661, Addgene) and the Cas9 expression vector (lentiCas9-Blast, #52962, Addgene), we generated an inducible Cas9 expression vector with a drug resistance gene switched (pCW-Cas9-Blast) through Gibson assembly.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Reversible Regulation of Polyubiquitin Gene UBC via Modified Inducible CRISPR/Cas9 System

doi: 10.3390/ijms20133168

Figure Lengend Snippet: Inducible dCas9–VP64 (idCas9–VP64) system for the upregulation of the polyubiquitin gene UBC and the UBC promoter region. ( A , B ) Human embryonic kidney 293T (HEK293T) cells were transiently transfected with idCas9–VP64 and then treated with 5 μg/mL doxycycline (Dox) for 1 day. Before harvest, cells were treated with 10 μM NaAsO 2 for 5 h. dCas9 and UBC mRNA levels were determined by qRT-PCR ( n = 3 each), normalized to GAPDH , and expressed as the fold change relative to the control (-Dox, -NaAsO 2 ). ( C ) Structure of the polyubiquitin gene UBC based on the NCBI gene reference and indication of the designed sgRNAs targeting the intron of UBC . All data are presented as means ± SEM from the indicated number of samples. *** p < 0.001 between two groups as indicated by horizontal bars. NS: Not significant.

Article Snippet: Using the catalytically inactive Cas9 (dCas9)-VP64 expression vector (pMSCV-LTR-dCas9–VP64, #46912, Addgene) and pCW-Cas9-Blast, we generated a Dox-inducible dCas9–VP64 expression vector (pCW-dCas9–VP64-Blast) through Gibson assembly.

Techniques: Transfection, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Reversible Regulation of Polyubiquitin Gene UBC via Modified Inducible CRISPR/Cas9 System

doi: 10.3390/ijms20133168

Figure Lengend Snippet: Evaluation of UBC upregulation using the idCas9–VP64 system with sgRNAs targeting the UBC intron. ( A ) HEK293T cells were transiently transfected with idCas9–VP64 and scrambled sgRNA and then treated with 5 μg/mL Dox for up to 2 days. dCas9 mRNA levels were determined by qRT-PCR ( n = 3 each), normalized to GAPDH , and expressed as the fold change relative to the control (0 days of Dox treatment). ( B ) HEK293T cells were transiently transfected with idCas9–VP64, and scrambed sgRNA (indicated as -) or sg UBC #1~#4, alone or in combination, and then treated with 5 μg/mL Dox for 2 days. dCas9–VP64 was detected by immunoblot analysis using an anti-Cas9 antibody. α-Tubulin was used as a loading control. ( C ) Expression of scrambled sgRNA (sgScramble) or sg UBC #1~#4 was determined by conventional RT-PCR. GAPDH was used as an internal control. ( D , E ) Transient transfection of HEK293T cells and Dox treatment were performed as described in ( B ). In ( E ), HEK293T cells were treated with 10 μg/mL Dox for 2 days. UBC mRNA levels were determined by qRT-PCR ( n = 3 each), normalized to GAPDH , and expressed as the fold change relative to the control (-Dox, scrambled sgRNA). Representative immunoblots and RT-PCR results are shown from three independent experiments ( B , C ). Data are presented as means ± SEM from the indicated number of samples ( A , D , E ). ** p < 0.01; *** p < 0.001 vs. control (0 days of Dox treatment) or between two groups as indicated by horizontal bars. NS: Not significant.

Article Snippet: Using the catalytically inactive Cas9 (dCas9)-VP64 expression vector (pMSCV-LTR-dCas9–VP64, #46912, Addgene) and pCW-Cas9-Blast, we generated a Dox-inducible dCas9–VP64 expression vector (pCW-dCas9–VP64-Blast) through Gibson assembly.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Reversible Regulation of Polyubiquitin Gene UBC via Modified Inducible CRISPR/Cas9 System

doi: 10.3390/ijms20133168

Figure Lengend Snippet: Application of an MS2 RNA aptamer and additional activators for UBC upregulation, based on the idCas9–VP64 system. ( A ) Schematic diagram of the working model of the MS2 aptamer and additional activators for the CRISPR activation system. MS2 protein conjugated to C-terminal activator domains, composed of the mouse NF-κB subunit (p65) and human heat shock factor 1 (HSF1), binds to the MS2 RNA stem-loop structure of sgRNA ver. 2. These sgRNA ver. 2 and MS2-p65-HSF1 complexes cooperate with idCas9–VP64 for the upregulation of target genes. ( B ) HEK293T cells were transiently transfected with idCas9–VP64, MS2-p65-HSF1, and scrambled sgRNA ver. 2 (indicated as -) or sg UBC #1~#4 ver. 2 (alone or in combination), and then treated with 10 μg/mL Dox for 2 days. UBC mRNA levels were determined by qRT-PCR ( n = 3 each), normalized to GAPDH , and expressed as the fold change relative to the control (-Dox, scrambled sgRNA ver. 2). ( C ) Transient transfection of HEK293T cells and Dox treatment were performed as described in ( B ). Ub conjugates (Ub n ) and free Ub were detected by immunoblot analysis using an anti-Ub antibody. α-Tubulin was used as a loading control. ( D , F ) HEK293T cells were transiently transfected with idCas9–VP64, MS2-p65-HSF1, and scrambled sgRNA (sgScramble) ver. 2 or sg UBC #3 ver. 2, and then treated with 10 μg/mL Dox for up to 4 days. To washout Dox, cells were treated with 10 μg/mL Dox for 2 days and it was then removed from the media for 2 days (2/-2). UBC and UBB mRNA levels were determined by qRT-PCR ( n = 3 each), normalized to GAPDH , and expressed as the fold change relative to the control (0 days of Dox treatment, scrambled sgRNA ver. 2). ( E ) HEK293T cells were transiently transfected with idCas9–VP64, MS2-p65-HSF1, and sg UBC #3 ver. 2, and then treated with 10 μg/mL Dox for up to 4 days. Dox washout (2/-2) was performed as described above. dCas9 mRNA levels were determined by qRT-PCR ( n = 3 each), normalized to GAPDH , and expressed as the fold change relative to the control (0 days of Dox treatment, scrambled sgRNA ver. 2). Representative immunoblots are shown from three independent experiments ( C ). All data are presented as means ± SEM from the indicated number of samples. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. control (0 days of Dox treatment or -Dox) or between two groups as indicated by horizontal bars. NS: Not significant.

Article Snippet: Using the catalytically inactive Cas9 (dCas9)-VP64 expression vector (pMSCV-LTR-dCas9–VP64, #46912, Addgene) and pCW-Cas9-Blast, we generated a Dox-inducible dCas9–VP64 expression vector (pCW-dCas9–VP64-Blast) through Gibson assembly.

Techniques: CRISPR, Activation Assay, Transfection, Quantitative RT-PCR, Western Blot