Cell-Penetrating Peptide-Mediated Delivery of TALEN Proteins via Bioconjugation for Genome Engineering

<div><p>Transcription activator-like (TAL) effector nucleases (TALENs) have enabled the introduction of targeted genetic alterations into a broad range of cell lines and organisms. These customizable nucleases are comprised of programmable sequence-specific DNA-binding modules derived from TAL effector proteins fused to the non-specific FokI cleavage domain. Delivery of these nucleases into cells has proven challenging as the large size and highly repetitive nature of the TAL effector DNA-binding domain precludes their incorporation into many types of viral vectors. Furthermore, viral and non-viral gene delivery methods carry the risk of insertional mutagenesis and have been shown to increase the off-target activity of site-specific nucleases. We previously demonstrated that direct delivery of zinc-finger nuclease proteins enables highly efficient gene knockout in a variety of mammalian cell types with reduced off-target effects. Here we show that conjugation of cell-penetrating poly-Arg peptides to a surface-exposed Cys residue present on each TAL effector repeat imparted cell-penetrating activity to purified TALEN proteins. These modifications are reversible under reducing conditions and enabled TALEN-mediated gene knockout of the human <i>CCR5</i> and <i>BMPR1A</i> genes at rates comparable to those achieved with transient transfection of TALEN expression vectors. These findings demonstrate that direct protein delivery, facilitated by conjugation of chemical functionalities onto the TALEN protein surface, is a promising alternative to current non-viral and viral-based methods for TALEN delivery into mammalian cells.</p></div

TALEN conjugation is reversible and R9 must be removed for TALEN cleavage activity.

<p>(A) Purified TALEN proteins are incubated with Cys-nitropyridyl (Npys) Arg₉ cell-penetrating peptide (R9-CPP) for 1 hr at room temperature. (B) <i>In vitro</i> 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.</p

Modification of the endogenous <i>BMPR1A</i> gene by cell-permeable TALEN proteins.

<p>(A) Frequency of gene disruption in HEK293 cells treated for 2 hr with 1.0 µM <i>BMPRIA</i>-targeting TALEN proteins conjugated at various peptide-to-protein ratios. All R9-conjugated <i>BMPR1A</i>-targeted TALEN proteins were labeled in the presence of protease inhibitor cocktail. (B) Comparison of the frequency of <i>BMPR1A</i> 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.</p

Regulation of Endogenous Human Gene Expression by Ligand-Inducible TALE Transcription Factors

The construction of increasingly sophisticated synthetic biological circuits is dependent on the development of extensible tools capable of providing specific control of gene expression in eukaryotic cells. Here, we describe a new class of synthetic transcription factors that activate gene expression in response to extracellular chemical stimuli. These inducible activators consist of customizable transcription activator-like effector (TALE) proteins combined with steroid hormone receptor ligand-binding domains. We demonstrate that these ligand-responsive TALE transcription factors allow for tunable and conditional control of gene activation and can be used to regulate the expression of endogenous genes in human cells. Since TALEs can be designed to recognize any contiguous DNA sequence, the conditional gene regulatory system described herein will enable the design of advanced synthetic gene networks

TAL effector structure.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085755#pone.0085755-Mak1" target="_blank">[41]</a>.</p

Enhancing the Specificity of Recombinase-Mediated Genome Engineering through Dimer Interface Redesign

Despite recent advances in genome engineering made possible by the emergence of site-specific endonucleases, there remains a need for tools capable of specifically delivering genetic payloads into the human genome. Hybrid recombinases based on activated catalytic domains derived from the resolvase/invertase family of serine recombinases fused to Cys₂-His₂ zinc-finger or TAL effector DNA-binding domains are a class of reagents capable of achieving this. The utility of these enzymes, however, has been constrained by their low overall targeting specificity, largely due to the formation of side-product homodimers capable of inducing off-target modifications. Here, we combine rational design and directed evolution to re-engineer the serine recombinase dimerization interface and generate a recombinase architecture that reduces formation of these undesirable homodimers by >500-fold. We show that these enhanced recombinases demonstrate substantially improved targeting specificity in mammalian cells and achieve rates of site-specific integration similar to those previously reported for site-specific nucleases. Additionally, we show that enhanced recombinases exhibit low toxicity and promote the delivery of the human coagulation factor IX and α-galactosidase genes into endogenous genomic loci with high specificity. These results provide a general means for improving hybrid recombinase specificity by protein engineering and illustrate the potential of these enzymes for basic research and therapeutic applications