CRISPR Screens Reveal Pig Genes Essential for African Swine Fever Infection

Using genome-wide CRISPR/Cas9 knockout screens to identified key host genes required for infection

African swine fever virus (ASFV) devastates pig health and global supply chains. To move beyond surveillance and culling, our engineering biology hub partnered with virology experts to run an unbiased, genome-wide CRISPR/Cas9 loss-of-function screen in porcine cells, revealing genes required by the virus for infection and taking the first steps toward ASFV resilient livestock.

Loss-of-function CRISPR/Cas9 screens are powerful tools to identify host factors required for virus entry and for downstream steps of viral replication. Finding “host dependency” genes creates actionable targets for breeding, gene editing, and therapeutics. This is of particular value in livestock species, where controlled challenge models and population genetics can rapidly translate discoveries into impact on farms.

 

Genetic insights

The CRISPR/Cas9 screen showed that MHC class II (SLA II) pathway genes as essential for productive ASFV infection, specifically the non-classical MHC II heterodimer SLA-DM (SLA-DMA/SLA-DMB), and upstream regulators RFXANK, RFXAP, and CIITA. Knocking out these genes nearly abolished viral replication. While re-expressing SLA-DM restored susceptibility, proving this host factor’s causal role. Ultimately, without SLA-DM, ASFV struggles to establish infection. Demonstrating how a virus can exploit the host’s immune machinery for its own replication.

 

From discovery to application

  • Genetic targets for resilience: SLA-DM and its regulators become concrete targets for precision breeding or editing strategies aimed at ASFV-resistant pigs.

  • A reusable discovery engine: The same genome-scale, loss-of-function screening workflow can interrogate entry factors and replication co-factors for other priority pathogens, or explore the genetic bases of production, welfare, and disease-tolerance traits in pigs and other livestock species.

     

Our infrastructure and expertise

  • Species-specific genome-wide libraries: We develop and deploy porcine and chicken CRISPR knockout libraries for unbiased functional genomics in livestock, closing a long-standing gap versus human/mouse toolkits.
  • High-throughput pooled screening: End-to-end design, lentiviral delivery at controlled MOI, iterative challenge models, and next-generation sequencing readout (MAGeCK analytics).
  • Rapid validation & mechanism: Follow-up gene knockout/knock-in lines to confirm causality and map where in the viral life cycle a host factor acts (e.g., SLA-DM in late endosomes during ASFV entry).
  • Translation pathways: We work with breeding and animal-health partners to prioritise safe, welfare-minded routes to disease resilience, from marker-assisted selection to carefully evaluated gene edits.
pig iPSC derived macs infected with ASFV
Pig iPSC-derived macrophages infected with ASFV. Cell nuclei are stained for DNA (blue), the viral early protein CP312R (green), and the late structural protein VP72 (red). Yellow indicates regions of colocalization between CP312R and VP72. CP312R is expressed early during infection, whereas VP72 expression marks late-stage infection when the virus is replicating and assembling within the cells. Images courtesy of Lynnette Goatley (Pirbright Institute) and Tom Watson (Roslin Institute).

 

A Collaborative Effort

This study was co-delivered with Friedrich-Loeffler-Institut (FLI) and the Roslin Institute, combining high-containment ASFV challenge expertise with our livestock CRISPR engineering platform. 

 

Key publication

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