Scientists Build Live African Swine Fever Virus From Synthetic DNA Using CRISPR

By Pooja Toshniwal PahariaReviewed by Lily Ramsey, LLMApr 2 2025

In a recent study published in Science Advances, researchers reported using synthetic genomics and a clustered regularly interspaced short palindromic repeats-­associated protein 9 (CRISPR-Cas9)–inhibited helper virus to create live African swine fever virus (ASFV) from lab-made deoxyribonucleic acid (DNA).

This method enables the rapid production of different virus mutants, potentially accelerating the development of treatments or vaccines—both subunit and live-attenuated—for ASF.

​​​​​​​Study: A synthetic genomics-based African swine fever virus engineering platform. Image Credit: CI Photos/Shutterstock.com

Introduction

ASF refers to a devastating illness affecting pigs and wild boars, causing severe organ damage and a 100% fatality rate. Initially identified in Kenya in 1921, ASF spread to Western Europe by the mid-20th century and has since spread to Asia, Papua New Guinea, and China.

The virus is enzootic in 26 sub-Saharan nations and has affected Haiti and the Dominican Republic since 2021. Live-attenuated African swine fever virus strains have demonstrated efficacy in Vietnam since 2022, resulting in licensed vaccines. However, recent studies suggest that these vaccines do not provide complete protection against all strains.

ASFV, a double-stranded DNA (dsDNA) virus belonging to the Asfarviridae family, causes African swine fever. The ASFV genome size varies, with more than 22 genotypes of ASFV identified. Researchers are developing effective and safe ASF vaccines using various methods, but the classical approach faces challenges in generating recombinant viruses.

Cas9 has improved recombination efficiency, but isolating mutant viruses remains time-consuming. Thus, more efficient technologies, such as synthetic genomics, are needed to identify molecular markers of ASFV pathogenesis and generate attenuated strains for vaccine development.

Synthetic biology-based reverse genetic approaches allow a high-throughput generation of genetically altered ASFV genomes. However, the non-infectious genome of ASFV complicates the application of reverse genetics.

Helper viruses are needed to reconstitute viruses with non-infectious genomes. Research communities have used helper viruses to reconstitute other viruses; however, this approach has not been applied to ASFV.

About the Study

In the present study, researchers applied helper virus-based synthetic genomics to rebuild live ASFV from the Kenya-IX-1033 strain of ASFV. They hypothesized booting up ASFV genomes by self-helper viruses (homologous or heterologous ASFV) whose replication is inhibited by Cas9 cleavage as far as the donor genome remains immune against the same cleavage activities.

The team generated mutants of genotype IX comprising single or multiple modifications across the ASFV genome. They modified the CD2v, K145R, A238L, and I329L genes using in vitro CRISPR-Cas9 editing.

They assembled the modified gene fragments to generate full-length ASFV Kenya-IX-1033 synthetic genomes through transformation-associated recombination (TAR) cloning in yeast and subsequent transfer to Escherichia coli.

These genomes contained gene deletions in the Cas9-resistant p12 mutant background. Polymerase chain reaction (PCR) amplification confirmed the genetic assemblies, further assessed by whole-genome sequencing (WGS) and Sanger sequencing. Synthetic hairpin loops enhanced genome assembly for reconstitution.

Further, researchers generated recombinant viruses containing C-terminal fusion proteins to facilitate functional characterization during infection by live-cell imaging. They assessed the plaque size of reconstituted ASFV strains from fluorescent images and performed growth kinetic assays using wild boar lung (WSL) cell lines.

Progeny viral titers were expressed in plaque-forming units (PFU) per mL of parental and reconstituted ASFV strains post-infection at a multiplicity of infection (MOI) value of 0.03.

The team used two recombinant viral strains, ASFV-NHVΔTK::GFP and SFVArmeniaΔ285L::GFPhuCD4, as self-helper viruses. Both strains release green fluorescent protein (GFP) and are inhibited by cleavage of the p30 gene by the Cas9 protein. The ΔCD2v::DsRed strain was considered the donor for ASFV reconstitution.

Results

WGS analysis showed that the reconstituted virus genome resembled the donor genome. The complete length of synthetic ASFV genomes assembled in yeast from overlapping gene fragments.

PCR and Sanger sequencing results indicated that the CP240L (p30) gene region and, partly, other loci (K146R and E199L) exhibited the wild-type Kenya-IX-1033 sequence.

Sequencing the genomes of reconstituted ASFV revealed heterologous helper–virus-derived sequences. The results confirm live virus reconstitution from transfected (synthetic) genomes.

The reconstituted virus exhibited growth kinetics and plaque sizes comparable to those of the parental strain. The synthetic genome was transfected into cells and infected with a helper virus.

In the experiments using helper viruses, whether heterologous or homologous reconstituting viruses from modified viral genomes, researchers observed recombination between the helper virus genome and the transfected genome at different locations.

However, homologous recombination shapes ASFV's genetic diversity, and the structure of the genomic termini is critical for ASFV replication.

Analysis of the resulting virus progenies from the green and double-fluorescent plaques revealed recombination between donor and helper virus genomes.

The results validate the hypothesis that self-helper viruses can reconstitute non-infectious genomes. The self-helper viruses were viable and grew without apparent defects, indicating their potential use in characterizing the functional role of CD2v and K145R during infection.

Implications and Future Outlook

The findings demonstrate the feasibility of reconstituting ASFV from synthetic genomes, facilitating further molecular research of ASFV and vaccine development.

Virus reconstitution could accelerate the development of vaccines for viruses with non-infectious genomes and rapidly generate engineering tools to develop countermeasures, especially for those with pandemic potential. This alleviates the considerable effort required to identify helper genes or helper viruses for live virus reconstitution.

Future studies may identify common transcribed genes for helper gene testing.  Nearly all ASFV mutants generated using reverse genetics grew at similar rates to wild-type viruses in vitro.

It would be intriguing to investigate whether these ASFV mutants attenuate in vivo and if they can protect against viral challenges.