Abstract:
Avian pathogenic Escherichia coli (APEC) are responsible for substantial morbidity and mortality in broiler chickens and have become a significant endemic disease in poultry industry worldwide. Lipopolysaccharides has been identified as an important virulence factor in E. coli and play an important role in APEC pathogenicity. The waaL gene encodes O-antigen ligase, which is known to mediate attachment of O-antigen to lipid A-core oligosaccharid and involve in mature LPS biosynthesis. There is an immense need to use new targeted therapies against infectious diseases. Antibiotics are the quick approach to treat APEC infection in poultry so far, but development of drug resistance strains due to the frequent use of antibiotics is making it a less acceptable option. Genome editing has revolutionized biological science by allowing scientists to alter the genomes for many purposes. CRISPR-Cas9 is the latest strategy for genome editing. In this study, in silico analysis verifies all physiochemical parameters based on the sequenced data of wild type and CRISPR/Cas9 mediated waaL gene knockout strains. The in silico analysis revealed that mutants are less hydrophobic (GRAVY index 0.534 and 0.380), less stable having instability index 37.97 in case of mutants than 10.87 in case of wild type, show greater variation in membrane composition and number of biofilm inhibitory peptides >63 than the wild type (BIP=63). In vivo models has been established and read-out system includes clinical symptoms & scores, mortality, postmortem analysis, pathomorphological changes in the heart, liver, and intestine, E. coli re-isolation for confirmation of APEC related pathogenesis. The in silico and in vivo results determine the success of the mutants generated by knocked out waaL gene using CRISPR-Cas9 reduced the level of infection caused by APEC. This approach can further be upgraded by knocking out more than one gene involved in causing APEC infection.
