One-Step Assembly of a PRRSV Infectious cDNA Clone and a Convenient CRISPR/Cas9-Based Gene-Editing Technology for Manipulation of PRRSV Genome

Abstract

Porcine reproductive and respiratory syndrome (PRRS) has been a persistent challenge for the swine industry for over three decades due to the lack of effective treatments and vaccines. Reverse genetics systems have been extensively employed to build rapid drug screening platforms and develop genetically engineered vaccines. Herein, we rescued recombinant PRRS virus (rPRRSV) WUH3 using an infectious cDNA clone of PRRSV WUH3 acquired through a BstXI-based one-step-assembly approach. The rPRRSV WUH3 and its parental PRRSV WUH3 share similar plaque sizes and multiple-step growth curves. Previously, gene-editing of viral genomes depends on appropriate restrictive endonucleases, which are arduous to select in some specific viral genes. Thus, we developed a restrictive endonucleases-free method based on CRISPR/Cas9 to edit the PRRSV genome. Using this method, we successfully inserted the exogenous gene (EGFP gene as an example) into the interval between ORF1b and ORF2a of the PRRSV genome to generate rPRRSV WUH3-EGFP, or precisely mutated the lysine (K) at position 150 of PRRSV nsp1α to glutamine (Q) to acquire rPRRSV WUH3 nsp1α-K150Q. Taken together, our study provides a rapid and convenient method for the development of genetically engineered vaccines against PRRSV and the study on the functions of PRRSV genes.

Keywords: CRISPR/Cas9; PRRSV; gene-editing; reverse genetics system.

Figure 1

Assembly of full-length infectious cDNA clone of PRRSV WUH3. (A) The PRRSV WUH3 genome was divided into six contiguous gene fragments (F1 to F6) based on the distribution of BstX I site. (B) Amplification of the six fragments of PRRSV WUH3. The gene fragments of PRRSV WUH3 were amplified using RT-PCR with specific primers. (C) Fusion of F1, the modified pCMV, and F6 to construct F6-pCMV-F1. (D) RFLP analysis of pCMV-WUH3 using BstXI.

Figure 2

Recovery, identification, and characterization of rPRRSV WUH3. (A) Expression of PRRSV-N protein in MARC-145 cells transfected with pCMV-WUH3 was detected by IFA at 5 days post-transfection. Additionally, cells were infected with PRRSV WUH3 (0.1 MOI), and PRRSV-N protein expression was assessed at 24 hpi. (B) The multiple-step growth curves of PRRSV WUH3 and rPRRSV WUH3 at an MOI of 0.1 were analyzed using TCID₅₀ assays. (C) Plaques formed by PRRSV WUH3 and rPRRSV WUH3 in MARC-145 cells were stained with 1% crystal violet at 96 hpi. (D) Areas of plaques formed by PRRSV WUH3 and rPRRSV WUH3 were detected using Image J and compared using GraphPad Prism 7. Data are displayed in a Violin Plot. ns, p > 0.5.

Figure 3

Insertion of the EGFP gene into PRRSV genome using CRISPR/Cas9. (A) Schematic diagram of modification of PRRSV genome by cleaving pCMV-WUH3 with CRISPR/Cas9 and subsequently ligating the EGFP gene into the cleaved pCMV-WUH3 through homologous recombination. (B) The cleavage of pCMV-WUH3 by CRISPR/Cas9 was examined using electrophoresis in 1% agarose gel. (C) The constructed pCMV-WUH3-EGFP was verified using Sanger sequencing.

Figure 4

Rescue and characteristics of rPRRSV WUH3-EGFP. (A) The expression of EGFP and PRRSV-N protein in MARC-145 cells transfected with pCMV-WUH3-EGFP or pCMV-WUH3 was examined using Western blotting assays. (B) MARC-145 cells were transfected with pCMV-WUH3-EGFP or pCMV-WUH3 for 5 days, the typical PRRSV CPE in MARC-145 cells and the green fluorescence were observed using a fluorescence microscope. (C) The multiple-step growth curves of rPRRSV WUH3-EGFP and rPRRSV WUH3 in MARC-145 at an MOI of 0.1 were analyzed using TCID₅₀ assays. (D) Plaques formed by rPRRSV WUH3-EGFP and rPRRSV WUH3 in MARC-145 cells were stained with 1% crystal violet at 96 hpi. (E) Areas of plaques formed by rPRRSV WUH3-EGFP and rPRRSV WUH3 were detected using Image J and compared using GraphPad Prism 7, respectively. Data are displayed in a Violin Plot. ns, p > 0.5.

Figure 5

The residue conservation analysis at the position of 150 in nsp1α of PRRSV isolates.

Figure 6

Site-specific mutation of PRRSV genome using CRISPR/Cas9. (A) Schematic diagram of mutating lysine at the position of 150 in nsp1α of PRRSV WUH3 using CRISPR/Cas9. (B) Confirmation of the constructed pCMV-WUH3 nsp1α-K150Q using Sanger sequencing. (C) The detection of rPRRSV WUH3 nsp1α-K150Q using IFA. MARC-145 cells were transfected with pCMV-WUH3 nsp1α-K150Q and pCMV-WUH3 for 5 days, followed by IFA to detect the expression of PRRSV-N protein. (D) Confirmation of the K150Q mutation in nsp1α of rPRRSV using Sanger sequencing. (E) The multiple-step growth curves of rPRRSV WUH3 nsp1α-K150Q and rPRRSV WUH3 in MARC-145 cells at an MOI of 0.1 were analyzed using TCID₅₀ assays. (F) Plaques formed by rPRRSV WUH3 nsp1α-K150Q and rPRRSV WUH3 in MARC-145 were stained with 1% crystal violet at 96 hpi. (G) Areas of plaques formed by rPRRSV WUH3 nsp1α-K150Q and rPRRSV WUH3 were detected using Image J and compared using GraphPad Prism 7, respectively. Data are displayed in a Violin Plot. ns, p > 0.5.