CRISPR/Cas9 Technology: Challenges and drawbacks

Genome engineering has been transformed in recent years by the introduction of the CRISPR technology for a variety of cancer research projects spanning from fundamental science to translational medicine and precision cancer treatment. Although there have been tremendous advancements in this area, a number of technical issues still need to be resolved, including off- target activity, inadequate indel or poor homology-directed repair (HDR) efficiency, in vivo distribution of the Cas system components, and immunological reactions. Chromosome rearrangements brought on by off-target effects might unintentionally affect some poorly matched genomic locations and restrict the use of CRISPR-Cas editing technologies for therapeutic reasons. Studies have shown that CRISPR-Cas tools may be more susceptible to off-target effects than some of the other common gene-editing techniques because a Cas protein is a monomer that might accidentally enhance the identification of shorter target sequences, whereas the TALEN and ZFN assemblies are dimeric. Off-target effects often come from Cas enzymes that cleave bystanders (not intended targets) and guide RNA to recognize mismatches. CRISPR systems delivered in vivo can trigger immune responses against foreign substances by significantly increasing people's innate immunity and/or adaptive immunity. Guide RNAs may be used to initiate innate immune responses. This article provides an overview of CRISPR-Cas applications from the lab to the clinic, identifies current barriers that may restrict the use of CRISPR-Cas systems as gene-editing toolkits in precision medicine, and offers some perspectives on how to address these barriers and speed up technical advancemen

Assessment of Phytochemicals, Antioxidant Activity and Enzyme Production of Endophytic Fungi Isolated from Medicinal Plant Sources

Endophytic fungi are mitosporic and meiosporic ascomycetes that asymptomatically reside in the internal tissues of plants beneath the epidermal cell layer, where fungi colonise healthy and living tissue via quiescent infections. Endophytes are important components of microbial diversity. Endophytic fungi isolated from medicinal plants more likely exhibit pharmaceutical potentials. These plentiful natural products isolated from endophytes represent a huge reservoir which offers an enormous potential for exploitation for medicinal, agricultural and industrial uses. There has been a great interest in endophytic fungi as potential producers of novel, biologically active products. Endophytes are believed to carry out a resistance mechanism to overcome pathogenic invasion by producing secondary metabolites. Globally, there are at least one million species of endophytic fungi in all plants which can potentially provide a variety of structurally unique natural products such as alkaloid, benzopyranones, chinones, flavanoids, phenols, steroids, xanthones and others. Therefore, there is an ample opportunity to unearth novel and interesting endophytic microorganisms with significant therapeutic efficacy. The objectives of our current study are to isolate endophytic fungi from specific medicinal plants found locally and carry out their characterization and isolation, followed by qualitative and quantitative assessment of secondary metabolites produced by them and study their antioxidant and enzyme activities. The present study, therefore would highlight the growing concept that the bioactive compounds produced by the endophytes not only establishes host endophyte relationship but also have an immense chance of application in the field of medicine, agriculture and industry

Strain-specific identification of Beauveria bassiana isolated from a novel habitat, using rDNA-based sequence analogy

Abstract Various branches of any living species are destined to specified identification. Henceforth, imposing of bioinformatics, including various supportive software along with wet lab experiments, has led to conclude genus and species of unidentified strains. Further, for the construction of phylogenetic trees, the principle of minimum evolution has been considered. Since few years, rapid sequencing of DNA has been adapted, and data on nucleotide sequences of genome in diverse organisms have accumulated at an accelerated pace. In the present study, molecular identification of the fungus, Beauveria bassiana, isolated from the soil of a tea cultivation area was endeavoured. Soil from Bogidhola tea garden of Golaghat District, Assam, India, has captured an attention as it holds some natural pesticidal properties. Since B. bassiana possesses substantial pesticidal property, an attempt was carried out to identify the presence of B. bassiana in that particular soil of tea garden, followed by its molecular identification. Sequencing of D2 subunit spanning 28S rDNA of isolated fungi had been completed for molecular identification imposing bioinformatic tools and among 15 isolates compared where a correction of 100% identification was observed with isolate of B. bassiana strain CCFC225554

Efficacy of a novel sequential enzymatic hydrolysis of lignocellulosic biomass and inhibition characteristics of monosugars

Efficient production of sugar monomers from lignocellulose is often hampered by serious bottle-necks in biomass hydrolysis. The present study reveals that ultra-sonication assisted pretreatment following autoclaving, termed as combined pretreatment, can lead to more efficient delignification of lignocellulosic biomass and an open, deformed polysaccharide matrix, found favorable for subsequent enzymatic hydrolysis, is formed. The pattern of inhibition for the enzymatic hydrolysis reaction on combined-pretreated saw dust is identified. Two main inhibition models (competitive and noncompetitive) are proposed and a better fit of experimental values with the theoretical values for the competitive inhibition model validates the proposition that in the present experiment, glucose inhibits the enzymes competitively. Additionally, accuracy of the inhibitory kinetics based models is estimated over a series of enzyme and substrate concentrations.acceptedVersio

Studies on delignification and inhibitory enzyme kinetics of alkaline peroxide pre-treated pine and deodar saw dust

Delignification of lignocellulosic biomass by alkaline peroxide pre-treatment is a preliminary important step for an overall biomass fractionation process. In the present work, saw dusts are pre-treated by aqueous alkaline peroxide solution under different temperatures over a predetermined time. It is seen that Combined Pre-treatment (CP) removes a substantially higher quantity of lignin from biomass under a particular temperature. At elevated temperatures, the extent of delignification is observed much better. The % removal is: [PR: 19.35%(30 °C):25.26%(50 °C):33.30%(100 °C)]; [CD:14.64%(30 °C):23.64%(50 °C): 28.83%(100 °C)]. Batch kinetics is investigated with certain models and corresponding parameters are estimated. As pre-treatment severity is strongly correlated to the pre-treatment temperature, increased value of “potential degree of delignification” is observed at escalated temperatures. Kinetics of enzymatic hydrolysis of delignified biomass shows decreased product inhibition with increased substrate concentration under a particular enzyme loading. Starting with a combination of 50 g/L substrate concentration with an enzyme loading of 13.23 g/L, an optimum concentration of 17.2 g/L and 21.19 g/L of glucose are produced from Pinus roxburghii and Cedrus deodara respectively. Experimental data fit quite well with the competitive inhibition kinetics based theoretical models with r2 ≥0.95. It is inferred that enzymes are competitively inhibited by glucose

Valorization of Lignocellulosic Waste (Crotalaria <i>juncea</i>) Using Alkaline Peroxide Pretreatment under Different Process Conditions: An Optimization Study on Separation of Lignin, Cellulose, and Hemicellulose

<p>Lignocellulose materials, essentially consisting of lignin, cellulose and hemicelluloses, are abundant sources of fermentable sugars. The bast fiber of Crotalaria <i>juncea</i> (<i>Sunn hemp</i>), a native cover crop of India, was used as feedstock for this study. The primary objective of this study was to add value to the waste C. <i>juncea</i> bast fiber. The same was achieved by pretreating the waste fiber using alkaline peroxide solution at various process conditions. The optimal process condition for alkaline peroxide pretreatment was identified for the fiber to pretreatment solution (S/S) ratio of 1/40 at 50°C for 5h with respect to all response variables (lignin removal, hemicellulose recovery, recovery of solid pretreated material, and crystallinity of lignocellulose). Three-way ANOVA results revealed that S/S ratio had no significant effect; whereas, both temperature and time, and the combination of parameters, exhibited significant effect on response variables. The characteristic peaks associated with lignin and cellulose demonstrated a higher amount of lignin removal and increased cellulose content with elevated treatment time. Autoclave assisted pretreatment proved to be inefficient due to removal of lower amount of lignin in addition to higher hemicellulose degradation. On the other hand, pretreatment using ultrasound was found to be most effective in removing lignin, liberating hemicelluloses along with diminition in cellulose crystallinity.</p

Efficacy of a novel sequential enzymatic hydrolysis of lignocellulosic biomass and inhibition characteristics of monosugars

Efficient production of sugar monomers from lignocellulose is often hampered by serious bottle-necks in biomass hydrolysis. The present study reveals that ultra-sonication assisted pretreatment following autoclaving, termed as combined pretreatment, can lead to more efficient delignification of lignocellulosic biomass and an open, deformed polysaccharide matrix, found favorable for subsequent enzymatic hydrolysis, is formed. The pattern of inhibition for the enzymatic hydrolysis reaction on combined-pretreated saw dust is identified. Two main inhibition models (competitive and noncompetitive) are proposed and a better fit of experimental values with the theoretical values for the competitive inhibition model validates the proposition that in the present experiment, glucose inhibits the enzymes competitively. Additionally, accuracy of the inhibitory kinetics based models is estimated over a series of enzyme and substrate concentrations