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Open AccessArticle

In Vivo Inhibition of Marek’s Disease Virus in Transgenic Chickens Expressing Cas9 and gRNA against ICP4

1
CSIRO Health and Biosecurity, Australian Centre for Disease Preparedness, Geelong, VIC 3220, Australia
2
Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
*
Author to whom correspondence should be addressed.
Current Address: CSIRO Health and Biosecurity, 5 Portarlington Road, Newcomb, VIC 3219, Australia.
Microorganisms 2021, 9(1), 164; https://doi.org/10.3390/microorganisms9010164
Received: 16 December 2020 / Revised: 7 January 2021 / Accepted: 11 January 2021 / Published: 13 January 2021
(This article belongs to the Special Issue Marek’s Disease Virus)
Marek’s disease (MD), caused by MD herpesvirus (MDV), is an economically important disease in chickens. The efficacy of the existing vaccines against evolving virulent stains may become limited and necessitates the development of novel antiviral strategies to protect poultry from MDV strains with increased virulence. The CRISPR/Cas9 system has emerged as a powerful genome editing tool providing an opportunity to develop antiviral strategies for the control of MDV infection. Here, we characterized Tol2 transposon constructs encoding Cas9 and guide RNAs (gRNAs) specific to the immediate early infected-cell polypeptide-4 (ICP4) of MDV. We generated transgenic chickens that constitutively express Cas9 and ICP4-gRNAs (gICP4) and challenged them via intraabdominal injection of MDV-1 Woodlands strain passage-19 (p19). Transgenic chickens expressing both gRNA/Cas9 had a significantly reduced replication of MDV in comparison to either transgenic Cas9-only or the wild-type (WT) chickens. We further confirmed that the designed gRNAs exhibited sequence-specific virus interference in transgenic chicken embryo fibroblast (CEF) expressing Cas9/gICP4 when infected with MDV but not with herpesvirus of turkeys (HVT). These results suggest that CRISPR/Cas9 can be used as an antiviral approach to control MDV infection in chickens, allowing HVT to be used as a vector for recombinant vaccines. View Full-Text
Keywords: Marek’s disease virus; CRISPR/Cas9; ICP4; transgenic chicken; disease resilience Marek’s disease virus; CRISPR/Cas9; ICP4; transgenic chicken; disease resilience
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MDPI and ACS Style

Challagulla, A.; Jenkins, K.A.; O’Neil, T.E.; Shi, S.; Morris, K.R.; Wise, T.G.; Paradkar, P.N.; Tizard, M.L.; Doran, T.J.; Schat, K.A. In Vivo Inhibition of Marek’s Disease Virus in Transgenic Chickens Expressing Cas9 and gRNA against ICP4. Microorganisms 2021, 9, 164.

AMA Style

Challagulla A, Jenkins KA, O’Neil TE, Shi S, Morris KR, Wise TG, Paradkar PN, Tizard ML, Doran TJ, Schat KA. In Vivo Inhibition of Marek’s Disease Virus in Transgenic Chickens Expressing Cas9 and gRNA against ICP4. Microorganisms. 2021; 9(1):164.

Chicago/Turabian Style

Challagulla, Arjun; Jenkins, Kristie A.; O’Neil, Terri E.; Shi, Shunning; Morris, Kirsten R.; Wise, Terry G.; Paradkar, Prasad N.; Tizard, Mark L.; Doran, Timothy J.; Schat, Karel A. 2021. "In Vivo Inhibition of Marek’s Disease Virus in Transgenic Chickens Expressing Cas9 and gRNA against ICP4" Microorganisms 9, no. 1: 164.

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