Chromosomal Translocations Detection in Cancer Cells Using Chromosomal Conformation Capture Data.

Complex chromosomal rearrangements such as translocations play a critical role in oncogenesis. Translocation detection is vital to decipher their biological role in activating cancer-associated mechanisms. High-throughput chromosomal conformations capture (Hi-C) data have shown promising progress in unveiling the genome variations in a disease condition. Until now, multiple structural data (Hi-C)-based methods are available that can detect translocations in cancer genomes. However, the consistency and specificity of Hi-C-based translocation results still need to be validated with conventional methods. This study used Hi-C data of cancerous cell lines, namely lung cancer (A549), Chronic Myelogenous Leukemia (K562), and Acute Monocytic Leukemia (THP-1), to detect the translocations. The results were cross-validated through whole-genome sequencing (WGS) and paired-read analysis. Moreover, PCR amplification validated the presence of translocated reads in different chromosomes. By integrating different data types, we showed that the results of Hi-C data are as reliable as WGS and can be utilized as an assistive method for detecting translocations in the diseased genome. Our findings support the utility of Hi-C technology to detect the translocations and study their effects on the three-dimensional architecture of the genome in cancer condition.

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.

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.