Bacterial Delivery of TALEN Proteins for Human Genome Editing

Abstract

Transcription Activator-Like Effector Nucleases (TALENs) are a novel class of sequence-specific nucleases that have recently gained prominence for its ease of production and high efficiency in genome editing. A TALEN pair recognizes specific DNA sequences and introduce double-strand break in the target site, triggering non-homologous end joining and homologous recombination. Current methods of TALEN delivery involves introduction of foreign genetic materials, such as plasmid DNA or mRNA, through transfection. Here, we show an alternative way of TALEN delivery, bacterial type III secretion system (T3SS) mediated direct injection of the TALEN proteins into human cells. Bacterially injected TALEN was shown to efficiently target host cell nucleus where it persists for almost 12 hours. Using a pair of TALENs targeting venus gene, such injected nuclear TALENs were shown functional in introducing DNA mutation in the target site. Interestingly, S-phase cells seem to show greater sensitivity to the TALEN mediated target gene modification. Accordingly, efficiency of such genome editing can easily be manipulated by the infection dose, number of repeated infections as well as enrichment of S phase cells. This work further extends the utility of T3SS in the delivery of functional proteins into mammalian cells to alter their characters for biomedical applications.

TALEN based HPV-E7 editing triggers necrotic cell death in cervical cancer cells

“…Researchers have focused on the use of type III secretion systems 39 and cell penetrating peptides 40 to transiently deliver TALENs and achieve desired effects. Therefore, in the context of HPV, TALENs could be locally delivered to that region and be given as adjuvant therapy along with surgery.…”

TALEN gene editing takes aim on HIV

“…For example, the Pseudomonas aeruginosa type III secretion system (injectosome) delivery of TALEN proteins into the nuclei of HeLa cells produces editing (Jia et al 2014). Likewise, liposomes and nanoparticles have been used to deliver anti-oncogenic drugs and may also show promise for delivering TALEN proteins to nuclei of cells (Malam et al 2009).…”

Engineered bacteria as therapeutic agents

“…An attenuated strain of Pseudomonas aeruginosa harboring a functional T3SS has been shown to deliver Cre-recombinase into the nucleus of mouse embryonic stem cells (or iPS) [62] triggering loxP mediated excision of the nuclear reprogramming cassette carrying c-Myc, Klf4, Oct4 and Sox2 [63]. The T3SS of this bacterial strain has also been used to translocate TALENs into human cells, which are capable of genome edition [64]. The same group reported the differentiation of mouse embryonic fibroblasts into myocites through the T3SS-mediated injection of MyoD, a transcription factor that acts as master regulator of myogenesis [65 ].…”