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.

SummaryTranslation elongation is modulated by various ribosome-binding proteins. Environmental stresses, such as starvation and antibiotics, can cause stalling of bacterial ribosomes, which may alter gene expression through a transcription or translation attenuation mechanism. In Pseudomonas aeruginosa, the expression of MexXY multidrug efflux system, which plays a significant role in resistance against aminoglycoside antibiotics, is controlled by a translation surveillance mechanism. Stalling of ribosome at the PA5471 leader peptide (PA5471.1) mRNA leads to transcription of PA5471, which subsequently up-regulates the expression of MexXY. In this study, we found that mutation in a suhB gene leads to decreased susceptibility to aminoglycosides. Transcriptomic analysis revealed an up-regulation of MexXY and PA5471, which were demonstrated to be responsible for the decreased susceptibility of the suhB mutant. We further demonstrated that PA5471.1 is essential for the up-regulation of PA5471 in the suhB mutant. Co-immunoprecipitation assay revealed an interaction between SuhB and ribosome, suggesting a role of SuhB in translation. Indeed, higher amount of PA5471.1 mRNA was found to associate with ribosome isolated from the suhB mutant, indicating increased ribosome stalling. Therefore, this study identified SuhB as a novel ribosome associated protein that is involved in modulating ribosome activity.