plasmids expressing 3 × grna  (GenScript corporation)

Journal: Theranostics

Article Title: The gRNA-miRNA-gRNA Ternary Cassette Combining CRISPR/Cas9 with RNAi Approach Strongly Inhibits Hepatitis B Virus Replication

doi: 10.7150/thno.18114

Figure Lengend Snippet: The effect of anti-HBV pri-miR-31-mimic flanking sequence in ternary cassette. ( A ) A dual-luciferase assay was conducted to detect the production of functional miR-HBV from gRNA3-miR-HBV-gRNA2 cassettes with different lengths of anti-HBV pri-miR-31 mimic in HuH7 cells co-transfected with pGL3-HBV (1575-1604) or pGL3-control, PRL-TK and each of the plasmids containing gRNA3-miR-HBV-gRNA2 cassettes with different length of anti-HBV pri-miR-31 mimics. The vectors pGL3-control and PRL-TK were used as negative control and internal control, respectively. Data was shown as mean±SD of 4 independent experiments. ( B ) A polyA tailing reaction and a quantitative reverse transcription-PCR (qRT-PCR) were conducted to detect the production of mature miR-HBV in HuH7 cells transfected with the plasmids containing gRNA3-miR-HBV-gRNA2 cassettes with different length of anti-HBV pri-miR-31 mimics. ( C ) Schematic illustration of gRNA-gRNA binary cassette. ( D ) The expression plasmid of 1.2×HBV was co-transfected with the expression plasmid containing 3-2 binary cassette or gRNA3-miR-HBV-gRNA2 ternary cassette with different length of anti-HBV pri-miR-31 mimic flanking sequence in HuH7 cells at the ratio of 3:1 and 1:3. HBsAg levels in the cell culture supernatant were measured using a time-resolved fluoroimmunoassay at 72 hours after transfection. Data was shown as mean±SD of 5 independent experiments. (* indicated P <0.05, Mann-Whitney U test). PX458 plasmid was used as a vector control. 5S rRNA was used as the internal control.

Article Snippet: The gRNA/Cas9 dual expression vector pSpCas9 (BB)-2A-GFP (PX458) was obtained from Addgene (Addgene, Cambridge, MA).

Techniques: Sequencing, Luciferase, Functional Assay, Transfection, Control, Negative Control, Reverse Transcription, Quantitative RT-PCR, Expressing, Plasmid Preparation, Cell Culture, MANN-WHITNEY

Journal: Theranostics

Article Title: The gRNA-miRNA-gRNA Ternary Cassette Combining CRISPR/Cas9 with RNAi Approach Strongly Inhibits Hepatitis B Virus Replication

doi: 10.7150/thno.18114

Figure Lengend Snippet: The function of miR-HBV in gRNA-miR-HBV-gRNA ternary cassettes. ( A ) A dual-luciferase assay was conducted to analyze the effect of 3-M38-2 ternary cassette in the target sequence of miR-HBV. The 3-M38-2 ternary cassette was used as a positive control. Data was shown as mean±SD of 5 independent experiments. ( B ) The expression plasmids of 1.2×HBV (genotype C) and miR-HBV or mutant miR-HBV (miR-HBVm) were co-transfected into HuH7 cells. The HBsAg levels in the cell and culture supernatant of HuH7 cells were measured using a time-resolved fluoroimmunoassay at 48 hours after transfection. Data was shown as mean±SD of 4 independent experiments. ( C ) The expression plasmids of 1.2×HBV and 3-2 binary cassette, 3-M38-3 or 3-H38-2 ternary cassette were co-transfected into HuH7 cells. The levels of HBsAg and HBeAg in the culture supernatant were measured using a time-resolved fluoroimmunoassay at 72 hours after transfection. Data was shown as mean±SD of 5 independent experiments. (D) The level of gRNA2 in the HuH7 cells transfected with the expression plasmid of vector control, 3-2 binary or 3-H38-2 ternary cassette was detected by qRT-PCR (SYBR Green). The level of (E) gRNA3 carrying flanking sequence of anti-HBV pri-miR-31 mimic and (F) mature miRNA (miR-HBV or mutant miR-HBV) in the HuH7 cells transfected with the expression plasmid of vector control, 3-2 binary or 3-H38-2 ternary cassette was detected by qRT-PCR (SYBR Green). Data was shown as mean±SD of 4 independent experiments. (** indicated P <0.01, ***indicated P <0.001, Mann-Whitney U test). PX458 plasmid was used as a vector control.

Article Snippet: The gRNA/Cas9 dual expression vector pSpCas9 (BB)-2A-GFP (PX458) was obtained from Addgene (Addgene, Cambridge, MA).

Techniques: Luciferase, Sequencing, Positive Control, Expressing, Mutagenesis, Transfection, Plasmid Preparation, Control, Quantitative RT-PCR, SYBR Green Assay, MANN-WHITNEY

Journal: Theranostics

Article Title: The gRNA-miRNA-gRNA Ternary Cassette Combining CRISPR/Cas9 with RNAi Approach Strongly Inhibits Hepatitis B Virus Replication

doi: 10.7150/thno.18114

Figure Lengend Snippet: The efficiency of gRNA-miR-HBV-gRNA ternary cassettes in suppressing HBV replication. The expression plasmids of each genotype (A, B or C) HBV and 3-H38-2 or 4-H38-1 cassette were co-transfected into HuH7 cells, and the levels of HBsAg ( A ) and HBeAg ( B ) in the cell culture supernatant or cells were measured using a time-resolved fluoroimmunoassay at 72 hours after transfection. Data was shown as mean±SD of 5 independent experiments. ( C ) The level of HBcAg in the HuH7 cells co-transfected with the expression plasmids of each genotype (A, B or C) HBV and 3-H38-2 or 4-H38-1 cassette were measured by Western Blot. The right figures were the statistical graphs of Western Blot, which was analyzed by Image J software (NIH, Bethesda, USA) at least in triplicate. HBc: HBcAg. PX458 plasmid was used as a vector control. (** indicated P<0.01, ***indicated P<0.001, Mann-Whitney U test).

Article Snippet: The gRNA/Cas9 dual expression vector pSpCas9 (BB)-2A-GFP (PX458) was obtained from Addgene (Addgene, Cambridge, MA).

Techniques: Expressing, Transfection, Cell Culture, Western Blot, Software, Plasmid Preparation, Control, MANN-WHITNEY

Journal: Theranostics

Article Title: The gRNA-miRNA-gRNA Ternary Cassette Combining CRISPR/Cas9 with RNAi Approach Strongly Inhibits Hepatitis B Virus Replication

doi: 10.7150/thno.18114

Figure Lengend Snippet: Detection on the HBV-specific gRNAs-mediated destruction of HBV genome. ( A ) The expression plasmids of 1.2×HBV and 3-2 binary, 3-M38-2, 3-H38-2 or 4-H38-1 ( B ) ternary cassette were co-transfected into HuH7 cells. Cellular DNA was extracted at 72 hours after transfection, and PCR amplifications were performed using the primers beyond the cleavage sites of two gRNAs. ( C ) Sequencing analysis of the smaller fragment cleft by gRNA2 and 3. ( D ) Sequencing analysis of the smaller fragment cleft by gRNA1 and 4. PX458 plasmid was used as a vector control.

Article Snippet: The gRNA/Cas9 dual expression vector pSpCas9 (BB)-2A-GFP (PX458) was obtained from Addgene (Addgene, Cambridge, MA).

Techniques: Expressing, Transfection, Sequencing, Plasmid Preparation, Control

Journal: Theranostics

Article Title: The gRNA-miRNA-gRNA Ternary Cassette Combining CRISPR/Cas9 with RNAi Approach Strongly Inhibits Hepatitis B Virus Replication

doi: 10.7150/thno.18114

Figure Lengend Snippet: Detection on the HBV-specific gRNAs-mediated destruction of HBV cccDNA. ( A ) HepAD38 cells were seeded into 10 cm dish. The expression plasmid of gRNA3-RNA2 binary or gRNA-miRNA-gRNA ternary cassette was co-transfected into HepAD38 cells. Transfection efficiency of HepAD38 cells was evaluated by observing the enhanced green fluorescent protein (EGFP) expressing cells under immunofluorescence microscopy. ( B ) HBsAg and HBeAg levels in the cell culture supernatant of HepAD38 cells were measured using a time-resolved fluoroimmunoassay. Data was shown as mean±SD of 6 independent experiments. ( C ) HBsAg levels in the cell culture supernatant of HepG2-NTCP-tet cells were measured using a time-resolved fluoroimmunoassay. Data was shown as mean±SD of 5 independent experiments. ( D ) HBV cccDNA levels in EGFP positive HepAD38 cells selected by Flow cytometry were measured using a KCl precipitation, plasmid-safe ATP-dependent DNase (PSAD) digestion, rolling circle amplification and quantitative PCR (SYBR green) combined method. Data was shown as mean±SD of 4 independent experiments. ( E ) PCR amplification of cccDNA was performed using the primers beyond the cleavage sites of two gRNAs following above rolling circle amplification. HepAD38 cells were transfected with 3-2 binary cassette expression plasmid, and then treated with or without lamivudine (3TC). The HBV DNA levels ( F ) in HepAD38 cells were detected by quantitative PCR, and HBV cccDNA levels ( G ) in EGFP positive HepAD38 cells selected by Flow cytometry were measured as above. Data was shown as mean±SD of 4 independent experiments. (* indicated P <0.05, ** indicated P <0.01, Mann-Whitney U test). PX458 plasmid was used as a vector control.

Article Snippet: The gRNA/Cas9 dual expression vector pSpCas9 (BB)-2A-GFP (PX458) was obtained from Addgene (Addgene, Cambridge, MA).

Techniques: Expressing, Plasmid Preparation, Transfection, Immunofluorescence, Microscopy, Cell Culture, Flow Cytometry, Amplification, Real-time Polymerase Chain Reaction, SYBR Green Assay, MANN-WHITNEY, Control