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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

Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster LA1 4YG, UK
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Author to whom correspondence should be addressed.
The authors contributed equally to this manuscript.
Vaccines 2019, 7(4), 207; https://doi.org/10.3390/vaccines7040207
Received: 14 November 2019 / Revised: 1 December 2019 / Accepted: 2 December 2019 / Published: 5 December 2019
(This article belongs to the Special Issue Herpesvirus Vaccines)
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. View Full-Text
Keywords: ILTV; vaccine; virus control; genome editing; Cre–lox; recombinant vaccines ILTV; vaccine; virus control; genome editing; Cre–lox; recombinant vaccines
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MDPI and ACS Style

Atasoy, M.O.; Rohaim, M.A.; Munir, M. 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 2019, 7, 207. https://doi.org/10.3390/vaccines7040207

AMA Style

Atasoy MO, Rohaim MA, Munir M. 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. 2019; 7(4):207. https://doi.org/10.3390/vaccines7040207

Chicago/Turabian Style

Atasoy, Mustafa O., Mohammed A. Rohaim, and Muhammad Munir. 2019. "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 7, no. 4: 207. https://doi.org/10.3390/vaccines7040207

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