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. 2020:2099:137-159.
doi: 10.1007/978-1-0716-0211-9_12.

Genetically Engineering a Susceptible Mouse Model for MERS-CoV-Induced Acute Respiratory Distress Syndrome

Sarah R Leist  1 , Adam S Cockrell  2
Affiliations

Genetically Engineering a Susceptible Mouse Model for MERS-CoV-Induced Acute Respiratory Distress Syndrome

Sarah R Leist et al. Methods Mol Biol. 2020.

Abstract

Since 2012, monthly cases of Middle East respiratory syndrome coronavirus (MERS-CoV) continue to cause severe respiratory disease that is fatal in ~35% of diagnosed individuals. The ongoing threat to global public health and the need for novel therapeutic countermeasures have driven the development of animal models that can reproducibly replicate the pathology associated with MERS-CoV in human infections. The inability of MERS-CoV to replicate in the respiratory tracts of mice, hamsters, and ferrets stymied initial attempts to generate small animal models. Identification of human dipeptidyl peptidase IV (hDPP4) as the receptor for MERS-CoV infection opened the door for genetic engineering of mice. Precise molecular engineering of mouse DPP4 (mDPP4) with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology maintained inherent expression profiles, and limited MERS-CoV susceptibility to tissues that naturally express mDPP4, notably the lower respiratory tract wherein MERS-CoV elicits severe pulmonary pathology. Here, we describe the generation of the 288-330+/+ MERS-CoV mouse model in which mice were made susceptible to MERS-CoV by modifying two amino acids on mDPP4 (A288 and T330), and the use of adaptive evolution to generate novel MERS-CoV isolates that cause fatal respiratory disease. The 288-330+/+ mice are currently being used to evaluate novel drug, antibody, and vaccine therapeutic countermeasures for MERS-CoV. The chapter starts with a historical perspective on the emergence of MERS-CoV and animal models evaluated for MERS-CoV pathogenesis, and then outlines the development of the 288-330+/+ mouse model, assays for assessing a MERS-CoV pulmonary infection in a mouse model, and describes some of the challenges associated with using genetically engineered mice.

Keywords: Cas9; Clustered regularly interspaced short palindromic repeats; Middle East respiratory syndrome coronavirus; Mouse; Pathogenesis.

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Figures

Fig. 1
Fig. 1
Timeline of the mammalian models evaluated for MERS-CoV pathogenesis between 2012 and 2019. Specific events since the emergence of MERS-CoV in 2012 are emphasized above the timeline. References to mammalian models evaluated for MERS-CoV pathogenesis comprise hamster [19], ferret [20], rabbit [–24], camel [–27], nonhuman primates [–35], and mouse [–48]
Fig. 2
Fig. 2
Comparison of DPP4 from different species. (a) Horizontal view of the crystal structure (PDB , 4L72) of human DPP4 (light gray) interacting with the MERS-CoV receptor binding domain (RBD; blue). The contact residues of human DPP4 with MERS-CoV RBD are highlighted in dark gray. (b) A 90° rotation, demonstrating the vertical view of (A). (c) Zoomed-in view of the human DPP4 structure (light gray) with highlighted MERS-CoV RBD contact residues (dark gray). Species-specific contact residues that differ from human are highlighted in red
Fig. 3
Fig. 3
CRISPR/Cas9 mediated genetic engineering of mouse DPP4. (a) In vitro validation of guide RNAs via Cas9 endonuclease assay (image was kindly provided by Dale Cowley in the Animal Models Core Facility at the University of North Carolina). Agarose gel separation based on size allows for discrimination between target DNA, Cas9 digested targets, and guide RNAs. (b) Schematic utilizing CRISPR/Cas9 technology to genetically engineer mice. Fertilized C57BL/6 J zygotes are collected and injected with RNA encoding Cas9, DPP4 single guide RNA, and oligos to facilitate homology-directed repair (HDR). Microinjected zygotes are implanted into pseudopregnant recipient female C57BL/6 J mice. Offspring are screened by sequencing for the intended change at positions 288 and 330. Mice identified as having the appropriate changes are backcrossed to C57BL/6 J mice to maintain the pure C57BL/6 J background, or may be crossed to any desired strain (e.g., BALB/cJ or 129S1/SvImJ). (c) Table describing sequences of Cas9 guide RNAs and oligos for HDR to genetically engineer amino acid changes at position 288 (Ala to Leu) and 330 (Thr to Arg). (d) Sequencing chromatograms highlighting how the F0 offspring from embryo implantation can be a mosaic of insertion/deletions (InDel’s) generated by random non-homologous end joining from Cas9 cutting at the genomic alleles, and the HDR repair that incorporates the intended changes encoding amino acids at positions 288 and 330. Pure homozygous 288–330+/+ lines were obtained by backcrossing onto C57BL/6 J mice. The highlighted mutations CAA (TTG in the reverse orientation) and AGA encode the novel 288 L and 330R amino acids
Fig. 4
Fig. 4
Mouse adaptation of MERS-0 in 288–330+/− mice. 288/330+/− mice were intranasally infected with 50 μL of MERS-0. Three days after infection lungs were harvested, homogenized in 1 mL PBS with glass beads, and 50 μL of the supernatant from the lung homogenate was used to infect the next round of 288–330+/− mice. Serial lung passages are performed for 15 [42] to 35 [44] rounds
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