Simultaneous Deletion of Virulence Factors and Insertion of Antigens into the Infectious Laryngotracheitis Virus Using NHEJ-CRISPR/Cas9 and Cre-Lox System for Construction of a Stable Vaccine Vector

Vaccines
Mustafa Ozan AtasoyMuhammad Munir

Abstract

Infectious laryngotracheitis virus (ILTV) is a promising vaccine vector due to its heterologous gene accommodation capabilities, low pathogenicity, and potential to induce cellular and humoral arms of immunity. Owing to these characteristics, different gene-deletion versions of ILTVs have been successfully deployed as a vector platform for the development of recombinant vaccines against multiple avian viruses using conventional recombination methods, which are tedious, time-demanding, and error-prone. Here, we applied a versatile, and customisable clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 accompanied with Cre-Lox system to simultaneously delete virulence factors and to insert foreign genes in the ILTV genome. Using this pipeline, we successfully deleted thymidine kinase (TK) and unique short 4 (US4) genes and inserted fusion (F) gene of the Newcastle disease virus without adversely affecting ILTV replication and expression of the F protein. Taken together, the proposed approach offers novel tools to attenuate (by deletion of virulence factor) and to generate multivalent (by insertion of heterologous genes) vaccine vectors to protect chickens against pathogens of poultry and public health importance.

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Citations

Aug 28, 2020·Vaccines·Mohammed A Rohaim, Muhammad Munir
Dec 18, 2020·Frontiers in Cellular and Infection Microbiology·Julianne VilelaMuhammad Munir
May 11, 2021·Frontiers in Cellular and Infection Microbiology·Ronghua DingGuangcai Duan
Sep 21, 2021·Applied Microbiology and Biotechnology·Rahul GuptaDipanjan Ghosh

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Methods Mentioned

BETA
genetic modification
PCR
confocal microscopy
electrophoresis
fluorescence microscopy

Software Mentioned

GraphPad Prism
GraphPad

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