CRISPR-Cas Systems: Programmable Nuclease Revolutionizing the Molecular Diagnosis.

CRISPR-Cas system has evolved as a highly preferred genetic engineering tool to perform target gene manipulation via alteration of the guide RNA (gRNA) sequence. The ability to recognize and cleave a specific target with high precision has led to its applicability in multiple frontiers pertaining to human health and medicine. From basic research focused on understanding the molecular basis of disease to translational approach leading to early and precise disease diagnosis as well as developing effective therapeutics, the CRISPR-Cas system has proved to be a quite versatile tool. The coupling of CRISPR-Cas mediated cleavage with isothermal amplification (ISA) of target DNA, followed by a read-out using fluorescent or colorimetric reporters appears quite promising in providing a solution to the urgent need for nucleic acid-based point-of-care diagnostic. Hence, it has been recognized as a highly sophisticated molecular diagnostic tool for the detection of disease-specific biomarkers not limited to nucleic acids-based detection but also of non-nucleic acid targets such as proteins, exosomes, and other small molecules. In this review, we have presented salient features and principles of class 2 type II, V, and VI CRISPR-Cas systems represented by Cas9, Cas12, and Cas13 endonucleases which are frequently used in molecular diagnosis. The article then highlights different medical diagnostic applications of CRISPR-Cas systems focusing on the diagnosis of SARS-CoV-2, Dengue, Mycobacterium tuberculosis, and Listeria monocytogenes. Lastly, we discuss existing obstacles and potential future pathways concerning this subject in a concise manner.

In silico study reveals unconventional interactions between MDC1 of DDR and Beclin-1 of autophagy.

DNA damage response (DDR) and autophagy are concerned with maintaining cellular homeostasis and dysregulation of these two pathways lead to pathologic conditions including tumorigenesis. Autophagy is activated as a protective mechanism during DDR which is indicative of their functional cooperativity but the molecular mechanism leading to the convergence of these two pathways during genotoxic stress remains elusive. In this study, through in silico analysis, we have shown an interaction between the Mediator of DNA damage checkpoint 1 (MDC1), an important DDR-associated protein, and Beclin-1, an autophagy inducer. MDC1 is an adaptor or scaffold protein known to regulate DDR, apoptosis, and cell cycle progression. While, Beclin-1 is involved in autophagosome nucleation and exhibits affinity for binding to Fork-head-associated domain (FHA) containing proteins. The FHA domain is commonly conserved in DDR-related proteins including MDC1. Through molecular docking, we have predicted the modeled complex between the MDC1 FHA domain and the Beclin-1 Coiled coil domain (CCD). The docking complex was modeled using ClusPro2.0, based on the crystal structure for the dimerized MDC1 FHA domain and Beclin-1 CCD. The complex stability and binding affinities were assessed using a Ramachandran plot, MD simulation, MM/GBSA, and PRODIGY webserver. Finally, the hot-spot residues at the interface were determined using computational alanine scanning by the DrugScorePPI webserver. Our analysis unveils significant interaction between MDC1 and Beclin-1, involving hydrogen bonds, non-bonded contacts, and salt bridges and indicates MDC1 possibly recruits Beclin-1 to the DSBs, as a consequence of which Beclin-1 is able to modulate DDR.