Designed gRNAs for CRISPR-Cas9 based antifungal resistance in eggplant.

Eggplant is an important vegetable crop and is a good source of antioxidants, minerals, and vitamins. It has been used in ancient medicines for the treatment of multiple diseases. However, the cultivated varieties of eggplant are susceptible to numerous pathogens and pests that have a negative impact on vegetable crops. Increased resistance achieved through resistance genes (R genes) is limited in eggplant breeding due to the fact that R genes are typically specific to a pathogen race and can be quickly surpassed by pathogen evolution. The susceptibility genes (S genes) in plants facilitate pathogen entry and proliferation, thus disabling these genes might be beneficial for providing a broad range and durable resistance against pathogens. Reports on crops such as Arabidopsis, rice, wheat, citrus, and tomatoes have highlighted that the knockout mutants of the S genes are tolerant to multiple different pathogens. The CRISPR/Cas9 system facilitates plant genome editing that can be utilized efficiently for crop improvement. In the current work, we have identified the homologs of candidate S genes DMR1, DMR6, EDR1, and PMR4/5/6 in the eggplant genome and designed and screened putative gRNAs against the identified target loci. The gRNAs were screened and selected on the basis of recognition of the PAM sequence, the MIT score, their minimum free energy, and the secondary structure. Five gRNAs for each gene homolog were selected after an in-depth analysis of all the predicted gRNAs using the above-mentioned criterion.

Microplastic properties and their interaction with hydrophobic organic contaminants: a review.

Microplastics (MPs) have been defined as particles of size < 5 mm and are characterized by hydrophobicity and large surface areas. MPs interact with co-occurring hydrophobic organic contaminants (HOCs) via sorption-desorption processes in aquatic and terrestrial environments. Ingestion of MPs by living organisms may increase exposure to HOC levels. The key mechanisms for the sorption of HOCs onto MPs are hydrophobic interaction, electrostatic interaction, π-π interactions, hydrogen bonding, and Van der Waals forces (vdW). Polymer type, UV-light-induced surface modifications, and the formation of oxygen-containing functional groups have a greater influence on electrostatic and hydrogen bonding interactions. In contrast, the formation of oxygen-containing functional groups negatively influences hydrophobic interaction. MP characteristics such as crystallinity, weathering, and surface morphology affect sorption capacity. Matrix properties such as pH, ionic strength, and dissolved organic matter (DOM) also influence sorption capacity by exerting synergistic/antagonistic effects. We reviewed the mechanisms of HOC sorption onto MPs and the polymer and matrix properties that influence the HOC sorption. Knowledge gaps and future research directions are outlined.

Towards the diagnosis of dengue virus and its serotypes using designed CRISPR/Cas13 gRNAs.

Dengue Virus (DENV) is a mosquito-borne virus that is prevalent in the world's tropical and subtropical regions. Therefore, early detection and surveillance can help in the management of this disease. Current diagnostic methods rely primarily on ELISA, PCR, and RT-PCR, among others, which can only be performed in specialized laboratories and require sophisticated instruments and technical expertise. CRISPR-based technologies on the other hand have field-deployable viral diagnostics capabilities that could be used in the development of point-of-care molecular diagnostics. The first step in the field of CRISPR-based virus diagnosis is to design and screen gRNAs for high efficiency and specificity. In the present study, we employed a bioinformatics approach to design and screen DENV CRISPR/Cas13 gRNAs for conserved and serotype-specific variable genomic regions in the DENV genome. We identified one gRNA sequence specific for each of the lncRNA and NS5 regions and identified one gRNA against each of DENV1, DENV2, DENV3, and DENV4 to distinguish the four DENV serotypes. These CRISPR/Cas13 gRNA sequences will be useful in diagnosing the dengue virus and its serotypes for in vitro validation and diagnostics.

Leaching characteristics and hazard evaluation of bottom ash generated from common biomedical waste incinerators.

India has more than 202 biomedical waste incinerators, however, knowledge on the chemical characteristics of incinerator ash is lacking. The objective of this study was to evaluate the lecahablility characteristics of bottom ash and to study the levels of incineration by-products viz. polycyclic aromatic hydrocarbons (PAHs), and polychlorinated biphenyls (PCBs). Bottom ash samples from 13 common biomedical waste treatment facilities (CBMWTF) were colleted and subjected to leachig test, sequential extraction procedure (SEP) and PAHs and PCBs analysis. Among metals, cadmium, chromium, manganese, lead and zinc were found higher than the regulatory limits indicating its hazardous nature. SEP showed that substantial fraction of Cd (30%) and Zn (25%) were associated with leachable fractions, whereas metals such as Cr, Fe, Mn, and Ni were mainly associated with reducible, organics and residual fractions. Concentrations of USEPA 16 priority PAHs ranged between 0.17-12.67 mg kg-1 and the total toxic equivalents (TEQ) were in the range of 0.9-421.9 ng TEQ/g. PAHs with 4-rings dominated all the samples and accounted for 68% to total PAHs concentrations. Concentration of Σ19 PCB congeners ranged from 420.4 to 724.3 µg kg-1. PCBs homologue pattern was dominated by mono- to tetra chlorinated congeners (60-86%). The findings indicate the need for segregation of plastics from biomedical waste, improvement of combustion efficiency, and efficient air pollution control devices for the existing incinerators in CBMWTFs.

Screening of CRISPR/Cas9 gRNA for mimicking Powdery Mildew resistant MLO ol-2 mutant.

Powdery Mildew (PM) caused by fungal pathogen Oidium neolycopersici (O. neolycopersici) affects both greenhouse and field-grown tomato production. Resistance to PM disease can be achieved by selective inactivation of Mildew Resistance Locus O (MLO) genes encoding heptahelical transmembrane domains, which confer susceptibility to fungal pathogens. Natural loss-of-function mutation is a 19 base pair (bp) deletion in the SlMLO1 gene locus responsible for fungal resistance in S. lycopersicum var. cerasiforme. Introgression of these resistance alleles through breeding into elite varieties is possible. However, this is a long and labour-intensive process and has limitations due to linkage drag. Nonetheless, recent developments in the field of genome editing technology particularly CRISPR/Cas9 systems allows quick, effective and accurate genome modification at the target gene locus. Therefore, it is of interest to determine the efficacy and exact deletion that mimics the natural ol-2 (Slmlo1) mutation present in wild tomatoes using CRISPR/Cas9. 947 putative guide RNAs (gRNAs) were designed using Cas9 variants to broaden Protospacer Adjacent Motif (PAM) compatibility and to enhance DNA specificity against the SlMLO1 locus. 60 out of 947 gRNAs were selected based on the recognition of the PAM sequence, the MIT specificity ranking, the off-target sites, their distance from the 19bp natural ol-2 mutation, the secondary structure of the gRNAs, and their minimum free energy. In depth analysis of these 60 gRNAs helped in the selection of the top five gRNAs based on the above-mentioned criteria. These gRNAs are useful for introducing deletions identical to natural ol-2 mutants and impart resistance against fungal pathogen O. neolycopersici in cultivated tomato crops.