CRISPR-Cas9, a tool to efficiently increase the development of recombinant African swine fever viruses

Abstract

African swine fever virus (ASFV) causes a highly contagious disease called African swine fever. This disease is often lethal for domestic pigs, causing extensive losses for the swine industry. ASFV is a large and complex double stranded DNA virus. Currently there is no commercially available treatment or vaccine to prevent this devastating disease. Development of recombinant ASFV for producing live-attenuated vaccines or studying the involvement of specific genes in virus virulence has relied on the relatively rare event of homologous recombination in primary swine macrophages, causing difficulty to purify the recombinant virus from the wild-type parental ASFV. Here we present the use of the CRISPR-Cas9 gene editing system as a more robust and efficient system to produce recombinant ASFVs. Using CRISPR-Cas9 a recombinant virus was efficiently developed by deleting the non-essential gene 8-DR from the genome of the highly virulent field strain Georgia07 using swine macrophages as cell substrate.

Figure 1

(A) Schematic representation of the 8DR gene deleted in ASFV-GΔ8-DR.CR and replaced with the RFP and blasticidin reporter gene cassette. (B) Sequence of the 8DR targeting gRNAs used to target the ASFV genome for editing.

Figure 2

Comparison of recombination efficiency of the two 8DR targeting gRNAs and traditional homologous recombination either by hemadsorption or by detection of RFP expressed as log₁₀ HAD₅₀/ml or log₁₀ TCID₅₀/ml. Frequency of recombination obtained with either technique is expressed as the ratio between the titers of the recombinant and the parental viruses.

Figure 3

Primary swine macrophages were inoculated with the indicated virus or mock infected, in the presence of red blood cells. In the first column bright field images were taken 20hpi, and observed for hemadsorption. In the second column RFP fluorescence was examined. The two images were merged in the third column.