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.

Keywords: ASFV; CRISPR/Cas9; gene editing; in-frame deletion; virus isolation.

FIGURE 1

CRISPR/Cas9-mediated genomic cleavage detection assay: (A) Schematic showing the gRNA-targeted region (red downward arrow) of the A238L (5EL) gene and the p3p4 primer pairs (flanking the gRNA target site) used to generate 499 bp fragment that would yield two distinct product bands of unequal sizes upon cleavage by the detection enzyme. (B) The 499 bp amplicon from analyzed clones (1–29) was spiked with similar amplicon from wildtype (w) ASFV to establish whether two additional distinct bands indicative of genomic cleavage are present. Clones labeled in blue produced mismatched amplicons hence are potentially edited. Clones labeled in green produced uniform amplicons (like wild-type amplicon) hence un-edited. (C) clones showing multiple bands in (B; labeled in blue) were subjected to further cleavage detection assay to rule out wildtype contamination. Clones (red) showing a single band were deemed to be edited clones. Numbers 1–29 indicates analyzed clones while w is the wildtype control.

FIGURE 2

Indels and frameshift mutations: (A) Amino acid sequences from edited clones show a consistently uniform 8 amino acid in-frame deletions at the cleavage and repair site in A238L (5EL). (B) Two-nucleotide insertion (shadowed red), 3nt upstream of the PAM (NGG) site generating a frame-shift (out-of-frame) mutation hence creating premature stop codons. The target location of the gRNA is shadowed in gray. (C) Multiple stop codons (red) and internal methionine codons (bold) generated in the frameshifted A238L (5EL) ORF by the 2nt insertion.

FIGURE 3

Schematic and confirmation of the A238L (5EL) knockout strategy in ASFV-Ke and ASFV-Ke-dsRed. (A) Linear PCR amplicon encoding enhanced green fluorescence protein (eGFP), under the control of p72 promoter and 10T thymidylate terminator, flanked by 1,000 bp-long regions homologous to the sequences upstream and downstream of A238L (5EL). Upon integration, the complete A238L (5EL) coding region is replaced with the eGFP cassette. Black arrows represent primers: p5 and p6 used to linearize the eGFP plasmid; p9, p10, p11, and p12 to confirm successful knockout of A238L; and p7 and p8 to confirm the absence of the A238L locus in ΔA238L viruses. p9 and p12 are external forward and reverse primers outside the recombination sites while p10 and p11, reverse and forward primers, respectively, are in-ternal to eGFP gene and are used in combination with external primers to confirm A238L (5EL) deletion. Amplification from primer pair p9/p10 confirms 5′ integration and amplification from primer pair p11/p12 confirms 3′ integration of eGFP cassette in the A238L (5EL) locus. The p9/p12 amplification discriminates ΔA238L (3.7 kb) from wildtype (3.3 kb) based on band sizes. (B) Agarose gel showing PCR screening using different primer combinations on ASFV-Ke-ΔA238L (1), ASFV-Ke-dsRedΔA238L (2), ASFV-Ke wt (3), ASFV-Ke-dsRed (4), and no-template control (5) confirming the successful integration of the eGFP cassette replacing the A238L (5EL) gene and showing that ΔA238L viruses are devoid of the wildtype A238L(5EL) gene. (C) Microscopic fluorescence images showing eGFP-expressing and dsRed-expressing ASFV-infected WSL cells.

FIGURE 4

In vitro replication kinetics of parental and mutant ASFVs: WSL cells were infected with the indicated viruses at an MOI of 0.1 and viral genome copies measured at 0, 4, 24, 48, 72, and 96 hpi. Data represent average of three independent infections and error bars represent the standard deviations.