Journal: Molecular Therapy. Nucleic Acids
Article Title: One-Vector System for Multiplexed CRISPR/Cas9 against Hepatitis B Virus cccDNA Utilizing High-Capacity Adenoviral Vectors
Figure Lengend Snippet: Proof of Concept of Anti-HBV Efficacy of TALENs or CRISPR/Cas9 (A) HEK293 cells were co-transfected with an HBV expression plasmid pTHBV2 and either of the complete nuclease systems. DNA was isolated after 4 days and examined for mutagenesis at target sites by T7E1 assay. Undigested and digested products were separated on an agarose gel side by side for comparison. Expected cleavage product sizes are indicated by arrowheads. “M” indicates the molecular weight marker lane. (B) Huh7 cells were co-transfected with an HBV expression plasmid pTHBV2 and TALEN or CRISPR/Cas9 nuclease systems. HBsAg concentrations in the supernatant were measured after 4 days by ELISA. Data are represented as means of S/CO (sample to control ratio) values and error bars indicate SD of three replicates. Statistically significant differences to HBV only are indicated by an asterisk (*p
Article Snippet: Mutation Detection For the T7 endonuclease 1 (T7E1) (New England Biolabs, MA) mutation detection assay, DNA sequences that span the target sites were amplified from purified genomic DNA by PCR using standard conditions for the OneTaq 2X Master Mix with Standard Buffer (New England Biolabs, MA), with the following primer sets: S/HBV-RT, S-T7 for 5′-ttcctcttcatcctgctgct-3′ and S rev 5′-tgtaaaaggggcagcaaaac-3′; X, No. 5-forw 5′-actcctagccgcttgttttg-3′ and No.5-rev 5′-ataagggtcgatgtccatgc-3′; C, C-T7 for 5′-ctgggtgggtgttaatttgg-3′ and No.6-rev 5′-tacccgccttccatagagtg-3′; P1, P1_F 5′-gctttcactttctcgccaac-3′ and P1_R 5′-accttgggcaatatttggtg-3′; and XCp, XCp_F 5′-actctctcgtccccttctcc-3′ and XCp_R 5′-gcctgagtgcagtatggtga-3′.
Techniques: TALENs, CRISPR, Transfection, Expressing, Plasmid Preparation, Isolation, Mutagenesis, Agarose Gel Electrophoresis, Molecular Weight, Marker, Enzyme-linked Immunosorbent Assay