In Vivo Inhibition of Marek's Disease Virus in Transgenic Chickens Expressing Cas9 and gRNA against ICP4

Abstract

Marek's disease (MD), caused by MD herpesvirus (MDV), is an economically important disease in chickens. The efficacy of the existing vaccines against evolving virulent stains may become limited and necessitates the development of novel antiviral strategies to protect poultry from MDV strains with increased virulence. The CRISPR/Cas9 system has emerged as a powerful genome editing tool providing an opportunity to develop antiviral strategies for the control of MDV infection. Here, we characterized Tol2 transposon constructs encoding Cas9 and guide RNAs (gRNAs) specific to the immediate early infected-cell polypeptide-4 (ICP4) of MDV. We generated transgenic chickens that constitutively express Cas9 and ICP4-gRNAs (gICP4) and challenged them via intraabdominal injection of MDV-1 Woodlands strain passage-19 (p19). Transgenic chickens expressing both gRNA/Cas9 had a significantly reduced replication of MDV in comparison to either transgenic Cas9-only or the wild-type (WT) chickens. We further confirmed that the designed gRNAs exhibited sequence-specific virus interference in transgenic chicken embryo fibroblast (CEF) expressing Cas9/gICP4 when infected with MDV but not with herpesvirus of turkeys (HVT). These results suggest that CRISPR/Cas9 can be used as an antiviral approach to control MDV infection in chickens, allowing HVT to be used as a vector for recombinant vaccines.

Keywords: CRISPR/Cas9; ICP4; Marek’s disease virus; disease resilience; transgenic chicken.

Figure 1

Schematic depiction of generating DF1 cells carrying the infected-cell polypeptide-4 (ICP4) gene fragments and GFP in a Tol2 transposon vector. Three stable DF1 cells carrying the ICP4 and GFP transgenes (DF1-pTIG#1, DF1-pTIG#2 and DF1-pTIG#3) were sorted after 2 weeks post-transfection.

Figure 2

Design and characterization of the guide RNAs (gRNAs) targeting ICP4 gene. (a) Schematic representation of six gRNAs designed to target the ICP4 gene. The PAM sequence was highlighted in red color. (b) Assessment of gRNAs against ICP4. DF1-pTIG#1, DF1-pTIG#2 and DF1-pTIG#3 cells were co-transfected with GFP-gRNA and a pair of ICP4-gRNAs in pX333 or an empty pX333 vector. Agarose gel electrophoresis of PCR products amplified using primers spanning across the targeted regions, respectively. Expected PCR amplicons: proximal DF1-pTIG#1 (wild-type (WT): ~400 bp; mutant: ~210 bp), middle DF1-pTIG#2 (WT: ~480 bp; mutant: ~300 bp) and distal DF1-pTIG#3 (WT: ~650 bp; mutant: ~400 bp). Targeted deletion of proximal (lane 2), middle (lane 3) and distal regions (lane 4 and 5) of ICP4 gene fragments in pTIG cells by corresponding ICP4 gRNAs in pX333 vector. Lanes 1 and 6, control DF1-pTIG#1 and DF1-pTIG#3 cells, respectively, transfected with empty pX333 vector. M, 1 kb + ladder. (c) Sequence comparison of WT and cleaved ICP4 PCR amplicons. The PAM is highlighted in red; the gRNA site is highlighted in blue and a base change is highlighted in green. (d) Effect of ICP4-gRNAs on MDV replication. Chicken embryo fibroblasts (CEF) were transfected with vectors carrying the Cas9 and ICP4 gRNAs (gICP4) or non-specific gRNAS (gNS) and infected with CVI988 MDV. Plaques were counted after four passages. The numbers of plaque-forming units (PFU) (on top of each column) for the cells transfected with ICP4-gRNAs (grey bar) are the average of two separate experiments and shown as the fraction of the PFU count for NS gRNAs (black bar), which is set at 1.0.

Figure 3

Generation of transgenic chickens and characterization of Cas9/gICP4 CEF. (a) Representative images of gonads harvested at stage 40 Hamburger Hamilton (HH) of injected embryos with the Tol2-gICP4-DsRed and pTrans complexed with L2000 CD complex. DsRed (right) and corresponding bright field (left) images of representative gonads. Fluorescent image of a hatched G1 gICP4-DsRed (b) and the Cas9-GFP (c) hen used to expand the Cas9-GFP line for this study. Hatched chicks were visually screened for GFP and DsRed expression using specific light sources for GFP or DsRed equipped in the goggles (BLS LTD, Hungary) and images were captured with a camera. (d) CEF were prepared from 10-day-old embryos obtained by breeding gICP4-DsRed and Cas9-GFP chickens. PCR genotyping of individual CEF cultures was performed using specific primers targeting the Cas9 transgene (top panel), gICP4 transgene (middle panel) or endogenous ovomucoid (OVM) gene (lower panel). Lane 1, gICP4 alone; lanes 2, 3, 8 and 12, Cas9/gICP4; lanes 4, 5, 6, 7, 9, 10, 11 and 13, WT; lane 14, water control. (e) Fluorescent microscopy images of CEF cultures #1, #2 and #4 expressing DsRed alone (gICP4 transgene alone), GFP+DsRed (Cas9/gICP4 transgenes) or no fluorescence (WT), respectively.

Figure 4

Specific inhibition of MDV and not herpesvirus of turkeys (HVT) in transgenic CEF. (a) Individual CEF cultures, Test (4 Cas9/gICP4) and control (total 9 CEF; 8 WT and 1 gRNA alone), were infected with 100 PFU of MDV Woodland (p19). Plaque numbers were calculated at 5 days post-infection (dpi). Error bars correspond to standard error of the mean (SEM). Statistical difference was calculated using a two-tailed Mann–Whitney test. p value indicates the level of significance between two groups. (b) The effect of the serotype 1-specific ICP4-gRNAs and Cas9 on MDV. CEF were prepared from WT (black bars) or Cas9/gICP4 (grey bars) embryos and plated at 2 × 10⁶ cells per well in a six-well plate. Triplicate cultures were infected with 100 PFU of serotype 1 MDV Woodland (p19) or serotype 3 HVT vaccine. The plaques were counted at 5 dpi. Values are shown as the mean of four replicates. Error bars correspond to SEM. p value indicates the level of significance between two groups. ns denotes not significant.

Figure 5

Inhibition of MDV in transgenic chickens expressing Cas9/gICP4. Transgenic chickens expressing Cas9/gICP4, Cas9 only or WT chickens were intraabdominally inoculated with MDV Woodland strain (p19). Spleens were harvested at (a) 6 and (b) 12 days post-infection (dpi) in experiment 1 and at (c) 33 dpi in experiment 2 for MDV isolation. Virus isolation assays were performed on splenocytes isolated from individual spleens by inoculating CEF with 5 × 10⁵ splenocytes/well in duplicate wells. PFU were counted at 5 dpi. The viral titers were shown as number of PFU per 5 × 10⁵ splenocytes. Error bars correspond to SEM. p value indicates the significance between two groups. ns denotes not significant. (d) qPCR-based verification of ICP4 DNA levels on 6-dpi experimental group chickens. y-axis: Delta Ct value; x-axis: PFU per 5 × 10⁵ splenocytes. Black dots: WT; green dots: Cas9-only; purple dots: Cas9/gICP4.