Journal: Scientific Reports

Article Title: Both TALENs and CRISPR/Cas9 directly target the HBB IVS2–654 (C > T) mutation in β-thalassemia-derived iPSCs

doi: 10.1038/srep12065

Figure Lengend Snippet: ( a ) Two pairs of TALENs and two sites of CRSIPR guide RNA were designed for in situ targeting of the HBB gene IVS2-654 mutation site. TALEN sites are indicated by blue lines, and guide RNA sites are indicated by red lines. The red nucleotide in the middle sequence is the IVS2-654 mutation site. PAM: protospacer adjacent motif. ( b ) Evaluation of TALEN- and CRISPR/Cas9-mediated DNA cleavage by a SSA (single strand annealing) assay. HEK293 cells were separately co-transfected with one of the pairs of TALENs or one of the pairs of CRISPRs and pSSA-HBB-IVS2 and TK-Renilla. At 48 h after transfection, the ratio of firefly luciferase and Renilla luciferase activity was measured by a microplate reader. TALEN-blank vector and pX330 blank vector were used as negative controls. The data represent the mean ± SD of three independent experiments. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0 .001) ( c ) Evaluation of TALEN- and CRISPR/Cas9-mediated DNA cleavage by the T7E1 assay in 293T cells. The endogenous locus was amplified by PCR, and the product was further purified according to the manufacturer’s instructions. The purified PCR product was denatured and reannealed, and the hybridized PCR products were further digested by T7 Endonuclease I. The upper lane shows the separation of the DNA on a 2% agarose gel, this results were cropped from the full-length gels which were presented in and all the gels were run under the same condition; while the lower lane shows relative indel rates in different groups as measured with the ImageJ program. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0 .001) ( d ) Evaluation of TALEN- and CRISPR/Cas9-mediated DNA cleavage by the T7E1 assay in 654hiPS cells. The upper lane shows the separation of DNA on a 2% agarose gel after T7E1 digestion; the lower lane shows the relative indel rates in different groups as measured by ImageJ. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0 .001) ( e ) Sanger sequencing of the different mutant types of HBB intron2 in 293T cells after transfection with either TALEN or CRISPR/Cas9. The blue nucleotides in the upper lane represent two pairs of TALEN recognition sites. The red nucleotides in the lower lane represent the PAM (protospacer adjacent motif) sequence recognized by CRISPR/Cas9.

Article Snippet: Luciferase activity was monitored with a microplate luminometer (Promega).

Techniques: TALENs, In Situ, Mutagenesis, Sequencing, CRISPR, SSA Assay, Transfection, Luciferase, Activity Assay, Plasmid Preparation, Amplification, Purification, Agarose Gel Electrophoresis