CRISPR-Based Gene Editing: a Modern Approach for Study and Treatment of Cancer.

The development and emergence of clustered regularly interspaced short palindromic repeats (CRISPR) as a genome-editing technology have created a plethora of opportunities in genetic engineering. The ability of sequence-specific addition or removal of DNA in an efficient and cost-effective manner has revolutionized modern research in the field of life science and healthcare. CRISPR is widely used as a genome engineering tool in clinical studies for observing gene expression and metabolic pathway regulations in detail. Even in the case of transgenic research and personalized gene manipulation studies, CRISPR-based technology is used extensively. To understand and even to correct the underlying genetic problem is of cancer, CRISPR-based technology can be used. Various kinds of work is going on throughout the world which are attempting to target different genes in order to discover novel and effective methodologies for the treatment of cancer. In this review, we provide a brief overview on the application of CRISPR gene editing technology in cancer treatment focusing on the key aspects of cancer screening, modelling and therapy techniques.

Purification, Characterization, and Application of Endoglucanase from Rhizopus oryzae as Antibiofilm Agent.

The enzyme endoglucanase is responsible for the depolymerization of cellulose. This study focuses on characterization and purification of endoglucanase from Rhizopus oryzae MTCC 9642 through a simple size exclusion method and its effective application as an antibiofilm agent. Extracellular ß-1,4-endoglucanase, an enzyme that catalyzes the hydrolysis of carboxymethyl cellulose, was found to be synthesized by Rhizopus oryzae MTCC 9642. The enzyme was purified up to homogeneity simply by size exclusion process through ultrafiltration and gel chromatography. The molecular weight of purified enzyme protein was estimated to be 39.8 kDa and it showed the highest substrate affinity towards carboxymethyl-cellulose with Km and Vmax values of 0.833 mg ml-1 and of 0.33 mmol glucose min-1 mg-1protein, respectively. The purified enzyme exhibited optimal activity at pH 6 with a broad stability range of pH 3-8. The most preferred temperature was 35 °C and 50% of activity could be retained after the thermal exposure at 40 °C for 25 min. The purified enzyme protein was inactivated by Cu2+, while the activity could be enhanced by the addition of exogenous thiols. Since biofilm is a challenge for health sector, with the aim of eradicating the biofilm, the purified endoglucanase was used to remove biofilm produced by two nosocomial bacteria. As predicted by in silico molecular docking interaction, the purified enzyme could effectively degrade biofilm architecture of bacterial strains S. aureus and P. aeruginosa by 76.52 ± 6.52% and 61.67 ± 8.76%, respectively. The properties of purified enzyme protein, as elucidated by in vitro and in silico characterization, may be favourable for its commercial applications as a potent antibiofilm agent.

Bio characterization of purified isoamylase from Rhizopus oryzae.

Extracellular isoamylase produced by Rhizopus oryzae PR7 MTCC 9642 in in Erlenmeyer flasks was purified by ultrafiltration and by two steps of Superose 6 C-10/300GL gel chromatography. The enzyme molecule was found to be a monomer with molecular weight of 68 kDa.The purified isoamylase showed optimum activity at pH 5.5 and temperature 55 °C. The catalytic activity was found to remain stable at a broad range of pH (4-8) and could show remarkable thermo resistance specially in presence of exogenous thiols. The noteworthy enhancement of activity in presence of Mn2+ indicated its role as enzyme cofactor while thermos and chemostability in presence of exogenous thiols indicated the presence of disulfide linkage at active site of the enzyme. Both in vitro study and doking analysis indicated the highest affinity of the isoamylase of R. oryzae PR7 toward glycogen and the enzyme exhibited Km and Vmax values of 0.38 mg/mL and 6.65 mM/min/mL, respectively. Purified debranching amylolytic enzyme from R. oryzae PR7 has potential for the study of glycogen and starch structure and industrial application in combination with other amylolytic enzymes. The rapid, convenient, relatively simple purification process and other functional attributes of the enzyme made it competent to be employed for industrial utilization.