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

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.

The African Swine Fever Isolate ASFV-Kenya-1033-IX is highly virulent and stable after growth in the wild boar cell line WSL

“…The copyright holder for this this version posted December 15, 2021. ; https://doi.org/10.1101/2021.12.15.472778 doi: bioRxiv preprint WSL compared to ASFV-Kenya-1033-IX grown in blood macrophages. The WSL grown ASFV-Kenya-1033-IX was used as a backbone for the introduction of gene modifications using CRISPR/Cas9 technology for the development of candidate liveattenuated vaccines (Abkallo et al 2021). Even after the development of candidate gene-deleted LA-ASFVs, which undergo additional passages to obtain pure clones, only few genomic changes were observed, indicating that the genome of ASF-Kenya-1033-IX is indeed very stable in WSL (Abkallo et al 2021).…”

“…The WSL grown ASFV-Kenya-1033-IX was used as a backbone for the introduction of gene modifications using CRISPR/Cas9 technology for the development of candidate liveattenuated vaccines (Abkallo et al 2021). Even after the development of candidate gene-deleted LA-ASFVs, which undergo additional passages to obtain pure clones, only few genomic changes were observed, indicating that the genome of ASF-Kenya-1033-IX is indeed very stable in WSL (Abkallo et al 2021). The few genomic changes could be random changes as mutations happen for any pathogen over generations or they could be due to adaptation to the cell line used for growth of the virus.…”

“…The copyright holder for this this version posted December 15, 2021. ; https://doi.org/10.1101/2021.12.15.472778 doi: bioRxiv preprint Whole-genome sequencing, genome assembly and Sanger sequencing DNA was extracted from the sucrose-purified virus using the Qiagen DNeasy blood and tissue kit (Qiagen) according to the manufacturer's protocol. Whole-genome sequencing was performed using the Illumina MiSeq platform at ILRI as described previously (Abkallo et al, 2021).…”

“…Sequence data generated for macrophage-grown and WSL-grown ASFV virus in this study were submitted to GenBank under SRA accessions SRR17226616 and SRR15187368 (Abkallo et al, 2021), respectively.…”

“…The most promising candidate vaccines are live-attenuated African swine fever viruses (LA-ASFV), which have shown to provide up to 100% protection against challenge with pathogenic ASFV (King et al ., 2011; O’Donnell et al ., 2015; Monteagudo et al ., 2017; Reis et al ., 2017; Borca et al ., 2020). The live-attenuated viruses show reduced virulence due to deletion of genes associated with virulence, either naturally (King et al ., 2011) by passage in tissue culture (Krug et al ., 2015; Rodríguez et al ., 2015) or by genetic modification techniques (Reis et al ., 2016; Borca et al ., 2018; Sánchez-Cordón et al ., 2018, Abkallo et al . 2021).…”