Rapid CRISPR/Cas9 Editing of Genotype IX African Swine Fever Virus Circulating in Eastern and Central Africa.

Hussein M Abkallo,Samuel O Oyola,Elias Awino,Walter Fuchs,Sanjay Vashee,Lucilla Steinaa,Bernard Oduor,Sonal P Henson,Stephen Mwalimu,Nicholas Svitek,Nacrya Assad-Garcia

doi:10.3389/fgene.2021.733674

Abstract

African swine fever virus (ASFV) is the etiological agent of a contagious and fatal disease of domestic pigs that has significant economic consequences for the global swine industry. Due to the lack of effective treatment and vaccines against African swine fever, there is an urgent need to leverage cutting-edge technologies and cost-effective approaches for generating and purifying recombinant virus to fast-track the development of live-attenuated ASFV vaccines. Here, we describe the use of the CRISPR/Cas9 gene editing and a cost-effective cloning system to produce recombinant ASFVs. Combining these approaches, we developed a recombinant virus lacking the non-essential gene A238L (5EL) in the highly virulent genotype IX ASFV (ASFV-Kenya-IX-1033) genome in less than 2 months as opposed to the standard homologous recombination with conventional purification techniques which takes up to 6 months on average. Our approach could therefore be a method of choice for less resourced laboratories in developing nations.

Highlights

African swine fever virus (ASFV) is a large, enveloped, double-stranded DNA virus, which is the etiological agent of a contagious and lethal disease of domestic pigs that has significant economic ramifications for the global swine industry
We chose the WSL cell because it is suitable for productive replication of ASFV (Portugal et al, 2012; Keil et al, 2014; Hübner et al, 2018), and it does not interfere with genome integrity of the investigated ASFV strain, whereas after adaptation of ASFV to Vero cells, large deletions resulting in loss of virulence and protective capabilities were frequently observed (Tabarés et al, 1987; Krug et al, 2015; Rodríguez et al, 2015)
We have provided a detailed description of the homology-directed repair (HDR) approach for editing the ASFV genome

Summary

Introduction

African swine fever virus (ASFV) is a large, enveloped, double-stranded DNA virus, which is the etiological agent of a contagious and lethal disease of domestic pigs that has significant economic ramifications for the global swine industry. The conventional technologies for producing ASFV mutants are based on homologous recombination involving replacement of viral genes with a reporter gene β-galactosidase (β-Gal), β-glucuronidase (GUS), or fluorescent markers (Rodríguez et al, 1992; Gómez-Puertas et al, 1995; Zsak et al, 1996; Neilan et al, 1997a; Afonso et al, 1998; Hernaez et al, 2006; Portugal et al, 2012; O’Donnell et al, 2015; Borca et al, 2017; Chen et al, 2020). There is an urgent need to deploy enabling technologies, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) (Barrangou et al, 2007) to expedite development of ASFV vaccines

Methods

Results

Conclusion