Simultaneous Deletion of Virulence Factors and Insertion of Antigens into the Infectious Laryngotracheitis Virus Using NHEJ-CRISPR/Cas9 and Cre–Lox System for Construction of a Stable Vaccine Vector

Figure 1. Homology-independent knocking-out of TK gene and knocking-in of a reporter gene into the infectious laryngotracheitis virus (ILTV) genome using CRISPR/Cas9-induced and non-homologous end-joining (NHEJ)-mediated double stranded DNA break and repair mechanisms. (A) Schematic diagram outlining the genomic organisation of the ILTV genome. The internal and terminal repeat short (IRS/TRS) and unique long (UL) and short (US) regions of the ILTV genome are shown. Important viral genes and their orientations are labelled at the bottom and approximate scale of the genome size is displayed at the top of the viral annotated genome. The schematic illustration of the donor plasmid and targeting strategy for CRISPR/Cas9-induced and NHEJ-mediated insertion of green fluorescent protein (GFP) reporter gene, which replaces the TK gene in the ILTV genome. The upstream promoter, downstream polyA tail, and sgRNA sites flanking the expression cassette are shown. (B) The leghorn male hepatoma (LMH) cells were transfected with plasmid carrying donor reporter cassette, expressing sg-A and Cas9 for 24 h and infection with ILTV until appearance of foci. Immunofluorescence visualisation of the ILTV plagues…

Figure 2. Homology-independent knock-out of US4 and knock-in of the F gene of Newcastle disease virus (NDV) into the ILTV genome using CRISPR/Cas9. (A) Schema of the ILTV and strategy for the construction of donor plasmid. The insertion cassette is shown in the inset. Two multiple cloning sites (MCS) were flanked upstream with the promoter and downstream with the corresponding polyA tail. Full length F gene of the NDV was cloned in the MCS1 site whereas dsRED was cloned in the MCS2 site. The dsRED cassette was flanked with the LoxP sites at the 5′ and 3′ ends to be excised by Cre-recombinase. The entire cassette was flanked with sgA/sgB target site to release the cassette by the Cas9 and was cloned between BsmB1 sites in the pcDNA3.1. The donor plasmid and plasmids expressing sgRNA targeting the entire length of the US4 and cassette, as well as Cas9 expression plasmids, were co-transfected in LMH cells for 24 h before infection with ΔTK-GFP+ ILTV. (B) The plagues co-expressing the GFP (representing ΔTK-GFP+ ILTV) and dsRED (representing ΔTK&ΔUS4-dsRED+ve ILTV) were visualised by confocal microscopy. (C) LMH cells, infected with ΔTK-GFP+ ILTV, ΔTK&ΔUS4-dsRED+ve ILTV, and wt ILTV viruses,…

Figure 3. Excision of dsRED from the ΔTK&ΔUS4-dsRED+ve ILTV using Cre–Lox system. (A) Genomic organisation of the ILTV and strategy for the Cre-recombinase-mediated excision of the dsRED through LoxP sites. (B) LMH cells were transfected with Cre-recombinase expressing plasmid or mock transfected for 24 h and were then infected with ΔTK&ΔUS4-dsRED+ve ILTV for 72 h. The excision of the dsRED was observed using fluorescence microscopy. Bright field, and dsRED and GFP-positive fields were presented. (C) LMH cells infected with wt ILTV, ΔTK-GFP+ ILTV, ΔTK&ΔUS4-dsRED+ve ILTV, and ΔTK&ΔUS4-Cre-ILTV viruses were collected for PCR analysis. Note the absence of dsRED in ΔTK&ΔUS4-Cre-ILTV-infected cells. (D) LHM cells were infected with ΔTK&ΔUS4-Cre-ILTV and stained for the F protein of NDV. (E) LMH cells were infected with ΔTK&ΔUS4-Cre-ILTV, and the expression of the GFP was visualised using fluorescence microscopy at different time points for 3 days.

Figure 4. Characterisation of the recombinant ILTVs generated using CRISPR/Cas9 and Cre–Lox system. (A) LMH cells were infected with wt ILTV, ΔTK-GFP+ ILTV, or ΔTK&ΔUS4-dsRED+ve ILTV (all passaged at least 15 times) or left uninfected for 48 h. Cells were lysed and expression of the F protein of NDV was determined using Western blotting, and alpha tubulin was used as loading control. (B) LMH cells were infected with wt ILTV, ΔTK-GFP+ ILTV, ΔTK&ΔUS4-dsRED+ve ILTV, ΔTK&ΔUS4-Cre-ILTV, or left uninfected for 48 h. Cells were lysed and expression of the F protein of NDV was determined using Western blotting, and alpha tubulin was used as loading control. (C) In vitro growth kinetics were performed using real-time PCR on DNA extracted from LMH cells at various time points post-infection with wt ILTV, ΔTK-GFP+ ILTV, ΔTK&ΔUS4-dsRED+ve ILTV, ΔTK&ΔUS4-Cre-ILTV, or from uninfected cells. (D) PCR was applied to confirm the stability of F gene expression cassette from the recombinant viruses at passage 1, 3, 5, 7, 10, and 15 in LMH cells using primers targeting the F gene. (E) LMH cells were left uninfected or infected with wt ILTV, ΔTK-GFP+ ILTV, ΔTK&ΔUS4-dsRED+ve ILTV, or ΔTK&ΔUS4-Cre-ILTV for 2 h…