T2A-linked TALENs demonstrate increased gene editing activity than co-transfected TALEN monomers. (a, d) Western blotting shows proper expression of co-transfected TALENs, tagged with 3xFLAG sequence, or pTAL10-TALENs, tagged with 2xHA/3xFLAG sequences, targeting MSTN or AAVS1 respectively. Asterisk (*) depicts uncleaved TALENs arms or GFP-TALEN. GAPDH was used as a loading control. (b, e) Representative gel of T7E1 assay comparing gene editing activities of co-transfected TALENs and T2A-linked TALENs targeting MSTN or AAVS1 respectively. Upper band, demarcated with an arrow, represents noncleaved DNA; bottom bands, demarcated with an arrow head, represent cleaved DNA fragments. Image is representative of 4–6 biological replicates. (c, f) Cleavage activity of TALEN (TALEN-L/R co-transfection vs. pTAL10-TALEN) targeting MSTN or AAVS1 respectively were compared using T7E1 endonuclease assay. Results are from 4–6 biological replicates. p < 0.05 was considered to be significant.