Figure 1.—

Structure and activity of TALE nucleases (TALENs). (A) Schematic of a transcription activator-like effector (TALE) protein. BamHI fragments were fused to the catalytic domain of the FokI endonuclease to create TALENs. TALEN activity was measured in an in vivo yeast assay that is described in the text (Townsend et al. 2009). N, N terminus; NLS, nuclear localization signal; B, BamHI site; AD, acidic activation domain. (B) Activity of TALENs constructed with TALEs AvrBs3 and PthXo1. Haploid cell types containing either TALEN expression or target plasmid in 200 μl of overnight culture were mated in YPD medium at 30°. After 4 hr, the YPD medium was replaced with 5 ml of selective medium and incubated overnight at 30°. Mated cultures were lysed, ONPG substrate added, and absorbance read at 415 nm using a 96-well plate reader (Townsend et al. 2009). β-Galactosidase levels were calculated as a function of substrate cleavage velocity and normalized to the ZFN positive control. Avr–FokI, AvrBs3 TALEN; Pth–FokI, PthXo1 TALEN; Avr–FokI and Pth–FokI, AvrBs3 and PthXo1 fusions to a catalytically inactive the version of FokI (Bitinaite et al. 1998); ZFN, zinc finger nuclease containing the Zif268 DNA binding domain as a positive control (Porteus and Baltimore 2003). (C) Activity of AvrBs3 and PthXo1 TALENS on targets with different spacer lengths. ZFN, Zif268-derived zinc finger nuclease. (D) Function of a heterodimeric TALEN. Activity in yeast containing PthXo1–FokI and AvrBs3–FokI expression vectors and a plasmid with a target consisting of recognition sites for each, in head-to-tail orientation separated by 15 bp is shown (Avr–FokI, Pth–FokI). Also shown for reference is activity of AvrBs3 (Avr–FokI) and PthXo1 (Pth–FokI) TALENS individually and Zif268 (ZFN) on their respective targets. As a negative control, a yeast culture with only the target site plasmid for Avr–FokI, Pth–FokI was assayed for LacZ activity [denoted as (−)].