RESUMEN
Burkholderia anthina is a pathogenic bacterial species belonging to the Burkholderiaceae family and it is mainly considered the etiological agent of chronic obstructive pulmonary diseases associated with cystic fibrosis, due to being intrinsic antibiotic resistant making it difficult to treat pulmonary infections. Hence increased rate of antibiotic-resistant bacterial species vaccine development is the priority to tackle this problem. In research work, we designed a multi-epitope-based vaccine construct against B. anthina using reverse vaccinology immunoinformatics and biophysical approaches. Based on the subtractive proteomic screening of core proteins we identified 3 probable antigenic proteins and good vaccine targets namely, type VI secretion system tube protein hcp Burkholderia, fimbria/pilus periplasmic chaperone and fimbrial biogenesis outer membrane usher protein. The selected 3 proteins were used for B and B cells B-derived T-cell epitopes prediction. In epitopes prediction, different epitopes were predicted with various lengths and percentile scores and subjected to further immunoinformatics analysis. In immunoinformatics screening a total number of 06, IDDGNANAL, KTVKPDPRY, SEVESGSAP, YGGDLTVEV, SVSHDTNGR, and GSKADGYQR epitopes were considered good vaccine target candidates and shortlisted for vaccine construct designing. The vaccine construct was designed by joining selected epitopes with the help of a GPGPG linker and additionally linked with cholera toxin b subunit adjuvant to increase the efficacy of the vaccine construct the sequence of the said adjuvant were retrieved from protein data bank through its (PDB ID: 5ELD). The designed vaccine construct was evaluated for its physiochemical properties analysis in which we reported that the vaccine construct comprises 216 amino acids with a molecular weight of 22.37499 kilo Dalton, 15.55 instability index (II) is computed, and this classifies that the vaccine construct is properly stable. VaxiJen v2.0 web server predicted that the vaccine construct is probable antigenic in nature with 0.6320 predicted value. Furthermore AllerTOP v. 2.0 tool predicted that the designed vaccine construct is non allergic in nature. Molecular docking analysis was done for analysis of the binding affinity of the vaccine construct with TLR-2 (PDB ID: 6NIG), the docking results predicted 799.2 kcal/mol binding energy score that represents the vaccine construct has a good binding ability with TLR-2. Moreover, molecular dynamic simulation analysis results revealed that the vaccine construct and immune cell receptor has proper binding stability over various environmental condition, i.e. change in pressure range, temperature, and motion. After each analysis, we observed that the vaccine construct is safe stable, and probably antigenic and could generate an immune response against the target pathogen but in the future, experimental analysis is still needed to verify in silico base results.
RESUMEN
The genome editing approach by clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) is a revolutionary advancement in genetic engineering. Owing to its simple design and powerful genome-editing capability, it offers a promising strategy for the treatment of different infectious, metabolic, and genetic diseases. The crystal structure of Streptococcus pyogenes Cas9 (SpCas9) in complex with sgRNA and its target DNA at 2.5 Å resolution reveals a groove accommodating sgRNA:DNA heteroduplex within a bilobate architecture with target recognition (REC) and nuclease (NUC) domains. The presence of a PAM is significantly required for target recognition, R-loop formation, and strand scission. Recently, the spatiotemporal control of CRISPR/Cas9 genome editing has been considerably improved by genetic, chemical, and physical regulatory strategies. The use of genetic modifiers anti-CRISPR proteins, cell-specific promoters, and histone acetyl transferases has uplifted the application of CRISPR/Cas9 as a future-generation genome editing tool. In addition, interventions by chemical control, small-molecule activators, oligonucleotide conjugates and bioresponsive delivery carriers have improved its application in other areas of biological fields. Furthermore, the intermediation of physical control by using heat-, light-, magnetism-, and ultrasound-responsive elements attached to this molecular tool has revolutionized genome editing further. These strategies significantly reduce CRISPR/Cas9's undesirable off-target effects. However, other undesirable effects still offer some challenges for comprehensive clinical translation using this genome-editing approach. In this review, we summarize recent advances in CRISPR/Cas9 structure, mechanistic action, and the role of small-molecule activators, inhibitors, promoters, and physical approaches. Finally, off-target measurement approaches, challenges, future prospects, and clinical applications are discussed.