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

Jia Liu,James T Patterson,Thomas Gaj,Carlos F Barbas Iii,Shannon J Sirk,Pierre-Antoine Defossez

doi:10.1371/journal.pone.0085755

Jia Liu, James T Patterson + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0085755

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Journal: PloS one	Publication Date: Jan 20, 2014
Citations: 229	License type: CC BY 4.0

Affiliation: Scripps Research Institute

Abstract

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 CCR5 and BMPR1A 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.

Highlights

Zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs) and CRISPR/Cas9-based systems are valuable reagents for inducing targeted genetic alterations within complex genomes [1,2]
In order to determine whether cell-penetrating peptides (CPPs)-TALEN proteins were capable of crossing cell membranes, we used Western blot to examine the lysate of HeLa cells treated with 2 mM of each CPP-TALEN protein
No TALENs was detected in any sample, indicating that cells do not take up CPP-TALENs from the surrounding medium (Fig. S1E)

Summary

Introduction

Zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs) and CRISPR/Cas9-based systems are valuable reagents for inducing targeted genetic alterations within complex genomes [1,2] These nucleases generate DNA double-strand breaks (DSBs) that can be repaired by error-prone non-homologous end joining (NHEJ) or homology-directed repair (HDR) [3]. A typical TAL effector consists of a series of 33- to 35-amino acid repeats that each recognizes a single base pair (bp) through two adjacent amino acid residues, termed the repeat variable di-residue (RVD) [22,23] This modularity allows creation of custom domains capable of recognizing virtually any DNA sequence [24], the repetitive structure of these units can result in rearrangements within TALEN genes when delivered into cells by lentiviral vectors [25]. Transfection of TALEN-encoding plasmid DNA or mRNA offers an alternative to viral-based methods, but these approaches are restricted to certain cell types and can be highly toxic [28]

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