Prediction and evaluation of multi epitope based sub-unit vaccine against Salmonella typhimurium.

Salmonella enteric serovar Typhimurium is the most common enteric pathogen in humans and animals. Consumption of contaminated food or water triggers inflammation that allows Salmonella to spread into the gut and causes gastrointestinal diseases. The infection spreads by intestinal invasion, phagocyte internalization and subsequent dissemination in many other patients. This research used TolA, a Salmonella typhimurium membrane protein, to computationally design a multi-epitope vaccine against the pathogen. Complete consistency of the candidate vaccine was checked In silico, and molecular dynamics simulations confirmed the vaccine's stability. According to docking report, the vaccine has a good affinity with toll-like receptors. In silico cloning and codon optimization techniques improved the vaccine's efficacy in Salmonella typhimurium manifestation process. The candidate vaccine induced an efficient immune response, as determined by In silico immune simulation. Computational studies revealed that the engineered multi-epitope vaccine is structurally stable, capable of eliciting particular immunological reactions, and therefore a candidate for a latent Salmonella typhimurium vaccine. However, wet lab studies and further investigations are required to confirm the results.

Reprogramming Mycobacterium tuberculosis CRISPR System for Gene Editing and Genome-wide RNA Interference Screening.

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), which is still the leading cause of mortality from a single infectious disease worldwide. The development of novel anti-TB drugs and vaccines is severely hampered by the complicated and time-consuming genetic manipulation techniques for M. tuberculosis. Here, we harnessed an endogenous type III-A CRISPR/Cas10 system of M. tuberculosis for efficient gene editing and RNA interference (RNAi). This simple and easy method only needs to transform a single mini-CRISPR array plasmid, thus avoiding the introduction of exogenous protein and minimizing proteotoxicity. We demonstrated that M. tuberculosis genes can be efficiently and specifically knocked in/out by this system as confirmed by DNA high-throughput sequencing. This system was further applied to single- and multiple-gene RNAi. Moreover, we successfully performed genome-wide RNAi screening to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be extensively used for exploring the functional genomics of M. tuberculosis and facilitate the development of novel anti-TB drugs and vaccines.

Treating Genetic Disorders Using State-Of-The-Art Technology.

CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated Protein 9), basically a bacterial immune system is now widely applicable to engineer genomes of a number of cells and organisms because of its simplicity and robustness. In research avenue the system has been optimized to regulate gene expression, modify epigenome and edit target locus. These applications make CRISPR/Cas9, a technology of choice to edit disease causing mutations as well as the epigenome more efficiently than ever before. Meanwhile its application in in vivo and ex vivo cells is encouraging the scientific community for more vigorous gene therapy and in clinical setups for therapeutic genome editing. Here we review the recent advances that CRISPR-Cas9 mediated genome editing has achieved and is reported in previous studies and address the challenges associated with it.

Improving CRISPR-Cas9 On-Target Specificity.

The CRISPR-Cas9 has revolutionized the field of molecular biology, medical genetics and medicine. The technology is robust, facile and simple to achieve genome targeting in cells and organisms. However, to propagate these nucleases for therapeutic application, the on-target specificity is of paramount importance. Although the binding and cleavage of off-target sites by Cas9 is issue of concern, however the specificity of CRISPR technology is greatly improved in current research employing the use of engineer nucleases, improved gRNA selection, novel Cas9 orhtologs and the advancement in methods to detect and screen off-target sites and its effects. Here we summarize the advances in this state-of-the-art technology that will equip the genome editing tools to be applied in clinical research. The researcher should optimize these methods with emphasize to achieve perfection in the specificity.

Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system.

Protein-protein interaction (PPI) network maintains proper function of all organisms. Simple high-throughput technologies are desperately needed to delineate the landscape of PPI networks. While recent state-of-the-art yeast two-hybrid (Y2H) systems improved screening efficiency, either individual colony isolation, library preparation arrays, gene barcoding or massive sequencing are still required. Here, we developed a recombination-based 'library vs library' Y2H system (RLL-Y2H), by which multi-library screening can be accomplished in a single pool without any individual treatment. This system is based on the phiC31 integrase-mediated integration between bait and prey plasmids. The integrated fragments were digested by MmeI and subjected to deep sequencing to decode the interaction matrix. We applied this system to decipher the trans-kingdom interactome between Mycobacterium tuberculosis and host cells and further identified Rv2427c interfering with the phagosome-lysosome fusion. This concept can also be applied to other systems to screen protein-RNA and protein-DNA interactions and delineate signaling landscape in cells.

Adaptive gene profiling of Mycobacterium tuberculosis during sub-lethal kanamycin exposure.

Resistance to anti-tuberculosis drugs is a formidable obstacle to effective tuberculosis (TB) treatment and prevention globally. New forms of multidrug, extensive drug and total drug resistance Mycobacterium tuberculosis (Mtb) causing a serious threat to human as well as animal's population. Mtb shows diverse adaptability under stress conditions especially antibiotic treatment, however underlying physiological mechanism remained elusive. In present study, we investigated Mtb's response and adaptation with reference to gene expression during sub-lethal kanamycin exposure. Mtb were cultured under sub-lethal drug and control conditions, where half were sub-cultured every 3-days to observe serial adaptation under same conditions and the remaining were subjected to RNA-seq. We identified 98 up-regulated and 198 down-regulated responsive genes compared to control through differential analysis, of which Ra1750 and Ra3160 were the most responsive genes. In adaptive analysis, we found Ra1750, Ra3160, Ra3161, Ra3893 and Ra2492 up-regulation at early stage and gradually showed low expression levels at the later stages of drug exposure. The adaptive expression of Ra1750, Ra3160 and Ra3161 were further confirmed by real time qPCR. These results suggested that these genes contributed in Mtb's physiological adaptation during sub-lethal kanamycin exposure. Our findings may aid to edify these potential targets for drug development against drug resistance tuberculosis.

Mycobacteria and Autophagy: Many Questions and Few Answers.

Tuberculosis (TB) is an ancient disease caused by Mycobacterium tuberculosis (Mtb). TB is one of the world's deadliest diseases, with one-third of infected individuals falling ill each year especially in many developing countries. Upon invading host cells, such as macrophages, Mtb can replicate in infected cells by arresting phagosome maturation and then potentially escaping into the cytosol. Host cells have a mechanism to control intracellular Mtb by inducing autophagy, which is an elaborate cellular process to target intracellular pathogens for degradation in infected cells. However, some factors of Mtb are involved in defense against killing by autophagy. Thus, this review highlights the recent advances in the interactions between autophagy and Mtb.

Keeping CRISPR/Cas on-Target.

CRISPR/Cas, a microbial adaptive immune system, has recently been reshaped as a versatile genome editing approach, endowing genome engineering with high efficiency and robustness. The DNA endonuclease Cas, a component of CRISPR system, is directed to specific target within genomes by guide RNA (gRNA) and performs gene editing function. However, the system is still in its infancy and facing enormous challenges such as off-target mutation. Lots of attempts have been made to overcome such off-targeting and proven to be effective. In this review we focused on recent progress of increasing the CRISPR specificity realized by rational design of gRNA and modification of Cas9 endonuclease. Meanwhile the methods to screen off-target mutation and their effects are also discussed. Comprehensive consideration and rational design to reduce off-target mutation and selection of effective screening assay will greatly facilitate to achieve successful CRISPR/Cas system mediated gene editing.