Question

Suppose you are working in the National Microbiology Laboratory in Winnipeg, Manitoba, the Canada’s only containment Level 4 facility where scientist can work safely with COVID-19 (SARS-CoV-2). At this stage you are permitted to perform your experiments in normal human bronchial epithelial (NHBE) cells and mouse models.

3a) To combat COVID-19, can you design a CRISPR/Cas strategy by targeting SARS-CoV-2 in NHBE cells. (Genebank accession number for SARS-CoV-2 genome sequence is NC_045512)

3b) It is now known that the spike protein of SARS-CoV-2 attaches to the host receptor angiotensin-converting enzyme 2 (ACE2), which allows the coronavirus to enter human cells and replicate. To combat COVID-19, one approach is to knockout the human ACE2 protein, another is to enhance ACE2’s expression in NHBE cells. Can you choose ONE approach (NOT both) and describe how you would do it? (Human ACE2 gene GenBank: AB046569).
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3c) It is debatable whether to use hydroxychloroquine (HCQ) to treat COVID-19 patients. To clarify it, could you design a single cell experiment to determine the transcriptome profiles of mice, which are infected with SARS-CoV-2 and treated with or without HCQ. (Note: mice (B6.Cg-Tg(K18-ACE2)2Prlmn/J) that express human ACE2 are from the Jackson Laboratory; CD4+ T cells, CD8+ T cells, B cells, and NK cells might be the focus).

Answer 1

3a) CRISPR-Cas9 is a genome editing tool which can be used effectively against SARS-CoV-2 by targeting the guide RNA in CRISPR-Cas9 system against essential genes of the virus such as the genes that code for spike protein or reverse transcriptase enzyme or proteins that assist in phage assembly inside the host cell.

3b) The best approach is to knockout human ACE2 receptor. As the viral entry is assisted by this receptor, overexpressing this will further enhance the severity of disease. ACE2 receptor can be knocked out using genome editing techniques such as CRISPR-Cas9 and gene silencing mechanisms such as RNAi. In both cases the guide RNA and small interfering RNA is to be made complementary to the ACE-2 receptor coding gene. This approach also has some side effects, since ACE2 receptor helps in maintaining angiotensin II homeostasis in the body and in transport of neutral amino acids in intestines.

3c) HCQ-Hydroxychloroquine is an antimalarial drug that affects the ACE2 receptor which is the entry point of SARS-CoV-2 virus. In this way it is assumed that it will lower the affinity of ACE2 receptor for Spike protein of SARS-Cov-2 virus. The transcriptome of two sets of mice treated with HCQ and without HCQ were analyzed quantitaviely for the presence of SARS-CoV-2 RNA. If the transcriptome from the HCQ treated mice contains higher amounts of SARS-CoV-2 RNA than the mice treated without HCQ then it will not be effective in treating COVID-19 with HCQ