Testing multiplexed anti-ASFV CRISPR-Cas9 in reducing African swine fever virus

Abstract

African swine fever (ASF) is a highly fatal viral disease that poses a significant threat to domestic pigs and wild boars globally. In our study, we aimed to explore the potential of a multiplexed CRISPR-Cas system in suppressing ASFV replication and infection. By engineering CRISPR-Cas systems to target nine specific loci within the ASFV genome, we observed a substantial reduction in viral replication in vitro. This reduction was achieved through the concerted action of both Type II and Type III RNA polymerase-guided gRNA expression. To further evaluate its anti-viral function in vivo, we developed a pig strain expressing the multiplexable CRISPR-Cas-gRNA via germline genome editing. These transgenic pigs exhibited normal health with continuous expression of the CRISPR-Cas-gRNA system, and a subset displayed latent viral replication and delayed infection. However, the CRISPR-Cas9-engineered pigs did not exhibit a survival advantage upon exposure to ASFV. To our knowledge, this study represents the first instance of a living organism engineered via germline editing to assess resistance to ASFV infection using a CRISPR-Cas system. Our findings contribute valuable insights to guide the future design of enhanced viral immunity strategies.

Importance: ASFV is currently a devastating disease with no effective vaccine or treatment available. Our study introduces a multiplexed CRISPR-Cas system targeting nine specific loci in the ASFV genome. This innovative approach successfully inhibits ASFV replication in vitro, and we have successfully engineered pig strains to express this anti-ASFV CRISPR-Cas system constitutively. Despite not observing survival advantages in these transgenic pigs upon ASFV challenges, we did note a delay in infection in some cases. To the best of our knowledge, this study constitutes the first example of a germline-edited animal with an anti-virus CRISPR-Cas system. These findings contribute to the advancement of future anti-viral strategies and the optimization of viral immunity technologies.

Keywords: African swine fever virus; CRISPR; agriculture; pig; xenotransplantation.

Fig 1

Multiplex CRISPR strategy targeting the ASFV genome to protect pig cells from ASFV infection. (a) DNA construct encoding six sgRNAs, each flanked by HH ribozyme and HDV ribozyme. Genes can be transcribed under one single promoter into a single RNA transcript that can be automatically processed into six mature sgRNA. The mature sgRNA assembles with Cas9 protein to form the catalytic active CRISPR complex. (b) sgRNA and Cas9 are constitutively expressed in the pig cells, cutting invading the ASFV genome either in the nucleus (route I) or cytoplasm (route II). The viral genome is subsequently degraded, and viral replication is stopped or attenuated. (c) Design illustration of the constructs for expressing both Cas9 protein and six sgRNAs. The transgenes are flanked by ITR sequences and inserted into the pig genome via Piggybac transposase. We tested Cas9 with and without the NLS signal. We also tested hEF1a and U6 promoter for 6-sgRNA expression.

Fig 2

Multiplex CRISPR strategy can efficiently cut ASFV DNA in vitro and different construct design has different effects on restricting ASFV replication in COS-7 cells. (a) Left panel, the single RNA transcript generated by IVT can be efficiently processed into monomeric gRNA with few dimeric (blue triangle) and trimeric gRNA. Right panel, the IVT single RNA transcript can mediate efficient cleavage of PCR amplicons amplified from the ASFV genome in vitro Cas9 cleavage assay. “DNA target #” indicates the PCR amplicon corresponding to the respective gRNA. The starting PCR amplicons (lane 01a, 03a, 04a, 05a, 06a, PCa) are digested into two or more fragments (red triangles, lane 01b, 03b, 04b, 05b, 06b, PCb. PC: positive control) after co-incubation with IVT single RNA transcript and Cas9 protein. (b) Engineered COS-7 single-cell clones with different gRNA promoters and Cas9 versions (left table) and relative Cas9 expression levels in those clones (right panel) determined by RT-qPCR. () ASFV replication in different COS-7 single-cell clones was measured by ASFV copy number in cell lysis using qPCR over 5 days. Strong inhibition of ASFV replication was only observed in COS-7 clones with high levels of Cas9 expression (GC49, GE22, GE64).

Fig 3

Production of transgenic pigs constitutively expressing Cas9 and 6 sgRNA targeting ASFV genome and virus challenge of the transgenic pigs. (a) Top table, the large white pig fibroblast single-cell clone GI58 harbors the pBv1-EF-6X construct with hEF1a promoter driving sgRNA and mEF1a promoter driving Cas9 with NLS. Bottom left panel, the presence of the transgene cassette was confirmed by PCR for the Cas9 gene in the genomic DNA of GI58. Bottom right panel, Cas9 expression was confirmed by RT-qPCR in the fibroblasts of the cloned pigs (GI58P10 and GI58P12) generated from GI58 fibroblasts. (b) photo of the cloned pigs generated from GI58 fibroblasts. (c) ASFV virus challenge design of the gene-edited pigs and wild-type pigs. Direct virus challenge was performed by muscular injection, while indirect virus challenge was performed by the cohabitation of the pigs in the same room with pigs under direct virus challenge. Each rectangle with four black squares indicates a separate room. In the light-red room, three gene-edited pigs were subjected to direct virus challenge, while in the light-gray room, three wild-type pigs were subjected to direct virus challenge. The additional one gene-edited pig and three wild-type pigs in each room are subjected to an indirect virus challenge. (d) Survival of the pigs in direct and indirect virus challenge. No statistical difference in survival time was observed in the two virus challenge modes comparing gene-edited pigs with wild-type pigs under the same condition. The arrow indicates indirect virus challenge, for example, W→G means wild-type pigs were under direct virus challenge and gene-edited pigs were under indirect virus challenge released by infected wildtype pigs. (e) ASFV titer in blood samples of the pigs in direct and indirect virus challenge measured by qPCR. Gene-edited pigs showed no detectable viral titer, much lower than wild-type pigs, in an indirect virus challenge released by infected gene-edited pigs (middle panel). No difference was observed in other conditions.