Figure 1.
(Left) Front view of the PthXo1 DNA-binding domain in the absence of target DNA and (right) side view in the presence of target DNA. Surface-exposed Cys residues depicted as white spheres. TAL effector repeats are colored cyan and purple. DNA is shown as grey sticks. PDB ID: 3UGM [41].
Figure 2.
TALEN conjugation is reversible and R9 must be removed for TALEN cleavage activity.
(A) Purified TALEN proteins are incubated with Cys-nitropyridyl (Npys) Arg9 cell-penetrating peptide (R9-CPP) for 1 hr at room temperature. (B) In vitro cleavage analysis of TALEN proteins conjugated at (left) various peptide-to-protein ratios and (right) various protein concentrations at a 30-to-1 peptide-to-protein ratio in the (top) absence or (bottom) presence of 10 mM DTT.
Figure 3.
Modification of the endogenous CCR5 gene by cell-permeable TALEN proteins.
(A) Frequency of endogenous gene disruption in HeLa cells treated with CCR5-targeting TALEN proteins conjugated to R9-CPPs at various (A) peptide-to-protein ratios, (B) TALEN concentrations (µM), and (C) incubation periods (hr). (D) Comparison of gene disruption frequencies in HeLa cells transfected with 200 ng TALEN expression vectors or treated with 1.0 µM TALEN proteins for 2 hr. Gene mutagenesis frequencies were determined by the Surveyor nuclease assay. All R9-conjugated CCR5-targeted TALEN proteins were labeled in the presence of protease inhibitor cocktail. (E) Sequence analysis of the CCR5 locus in HeLa cells treated with 1.0 µM conjugated TALEN proteins for 2 hr. Deletions (dashes) and insertions (lowercase) induced by NHEJ repair are aligned to the wild-type sequence (WT). Black triangles indicate expected Surveyor nuclease assay cleavage products.
Figure 4.
Modification of the endogenous BMPR1A gene by cell-permeable TALEN proteins.
(A) Frequency of gene disruption in HEK293 cells treated for 2 hr with 1.0 µM BMPRIA-targeting TALEN proteins conjugated at various peptide-to-protein ratios. All R9-conjugated BMPR1A-targeted TALEN proteins were labeled in the presence of protease inhibitor cocktail. (B) Comparison of the frequency of BMPR1A knockout in HEK293 cells transfected with 200 ng of TALEN expression vectors or treated with 1.0 µM cell-permeable TALEN proteins for 2 hr. Black triangles indicate expected Surveyor nuclease assay cleavage products.