Efficient Gene Editing in Pluripotent Stem Cells by Bacterial Injection of Transcription Activator-Like Effector Nuclease Proteins

Abstract

The type III secretion system (T3SS) of Pseudomonas aeruginosa is a powerful tool for direct protein delivery into mammalian cells and has successfully been used to deliver various exogenous proteins into mammalian cells. In the present study, transcription activator-like effector nuclease (TALEN) proteins have been efficiently delivered using the P. aeruginosa T3SS into mouse embryonic stem cells (mESCs), human ESCs (hESCs), and human induced pluripotent stem cells (hiPSCs) for genome editing. This bacterial delivery system offers an alternative method of TALEN delivery that is highly efficient in cleavage of the chromosomal target and presumably safer by avoiding plasmid DNA introduction. We combined the method of bacterial T3SS-mediated TALEN protein injection and transfection of an oligonucleotide template to effectively generate precise genetic modifications in the stem cells. Initially, we efficiently edited a single-base in the gfp gene of a mESC line to silence green fluorescent protein (GFP) production. The resulting GFP-negative mESC was cloned from a single cell and subsequently mutated back to a GFP-positive mESC line. Using the same approach, the gfp gene was also effectively knocked out in hESCs. In addition, a defined single-base edition was effectively introduced into the X-chromosome-linked HPRT1 gene in hiPSCs, generating an in vitro model of Lesch-Nyhan syndrome. T3SS-mediated TALEN protein delivery provides a highly efficient alternative for introducing precise gene editing within pluripotent stem cells for the purpose of disease genotype-phenotype relationship studies and cellular replacement therapies.

High‐efficiency protein delivery into transfection‐recalcitrant cell types

“…Previously, we constructed an attenuated P. aeruginosa strain named ∆8 which showed high T3SS activity with low cytotoxicity (Neeld et al, ; Table S1). However, a high dose of antibiotics had to be used to eliminate resident bacterial cells after the protein delivery (Bai et al, ; Jia et al, ; Jin et al, ). Application of antibiotics not only increases the cost but also restricts its application due to possible selection of antibiotic‐resistant variants, therefore, use of the auxotrophic mutant is a good strategy to achieve self‐elimination without the use of antibiotics.…”

Bacterial type III secretion system as a protein delivery tool for a broad range of biomedical applications

“…An attenuated P. aeruginosa strain Δ8, deleted of 7 virulence-related genes (exoS/T/Y, ndk, xcpQ, lasI, rhlI) and one T3S suppressor gene (popN), was employed as the protein delivery vector (Bai et al, 2015;Jia et al, 2015;Neeld et al, 2014). Proteins of interest were cloned and expressed on an Escherichia-Pseudomonas shuttle expression plasmid (Fig.…”

“…Further, in combination with a single-stranded oligonucleotide DNA template, the T3SS injected TALENs effectively introduced a single nucleotide change in the GFP gene via homologous recombination, converting a "GAG" to a "TAG" stop codon, thus switching off the GFP. This approach resulted in a higher efficiency of target site modification (~3 folds) than the conventional transfection method (Jia et al, 2015).…”

Rationale redesign of type III secretion systems: toward the development of non-pathogenic E. coli for in vivo delivery of therapeutic payloads

“…Following the initial establishment of T3SSs as antigen delivery systems, they were demonstrated to be capable of injecting a variety of proteins of therapeutic value, including anti-inflammatory cytokines [22], camelid-derived single domain antibodies [23,24] and transcription activator-like effector nucleases (TALENs) [25,26]. Furthermore, the Jin lab described protocols for how the repeated delivery of mammalian reprogramming factors via the T3SS of an attenuated effector-less Pseudomonas strain can be used to promote the transdifferentiation of mouse embryonic fibroblasts into myocytes [27] and murine embryonic stem cells into cardiomyocytes [28]; thus, expanding upon the potential clinical applications of repurposed T3SSs.…”