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.
Highlights
Transcription activator-like (TAL) effectors were first discovered in plant pathogen Xanthomonas sp. which directly injects the TAL effectors into plant cells through a type III secretion system (T3SS), where the TAL effectors bind to and regulate plant genes to facilitate the bacterial colonization [1]
To deliver Transcription Activator-Like Effector Nucleases (TALENs) proteins using bacterial T3SS, the two TALENs were cloned into pExoS54F-TAL where a bacterial promoter with N-terminal 54 amino acids of ExoS were fused to the TALEN with a FLAG-tag in the fusion junction (Fig. 1B)
TALEN technology is a powerful tool in creating knockouts in various eukaryotic model organisms, the TALENs are currently delivered into the host cells in the form of plasmid DNA or RNA, posing the potential danger of exogenous genetic materials integrating into the genome of target cells
Summary
Transcription activator-like (TAL) effectors were first discovered in plant pathogen Xanthomonas sp. which directly injects the TAL effectors into plant cells through a type III secretion system (T3SS), where the TAL effectors bind to and regulate plant genes to facilitate the bacterial colonization [1]. Transcription activator-like (TAL) effectors were first discovered in plant pathogen Xanthomonas sp. The DNA binding domain can be assembled using four types of repeats that recognize corresponding four nucleotides [2,3,4]. A novel class of sequence-specific nucleases have been generated by fusing the TAL effector to the catalytic domain of FokI endonuclease, called Transcription Activator-Like Effector Nucleases (TALENs) [5,6]. TALENs are composed of a DNA binding domain that is capable of directing the FokI nuclease to a specific target site. The TALEN induced DSBs activate the DNA repair system within cells which stimulates ‘‘non-homologous end joining’’ in the absence of homologous DNA template. In the presence of a homologous template DNA, the DSBs triggers homologous recombination, introducing desired DNA sequence alterations
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