Question

You just got hired as a research associate in a laboratory that studies coronaviruses. SARS-CoV and SARS-CoV-2 both use the Angiotensin Converting Enzyme-2 (ACE2) protein as a receptor. Binding of these viruses to ACE2 triggers viral entry into the host cell. Your goal is to use CRISPR/Cas9 genome editing to knockout expression of ACE2 to prevent viral entry into cultured lung cells

Let’s assume you’ve successfully knocked out ACE2 expression. What is the next experiment you’d like to perform?

Answer 1

The next experiment after knocking out ACE2 in cultured lung cells would be to test the cells' susceptibility to coronavirus infection and observe the effects on viral entry and replication.

Step-by-step explanation:

After successfully using CRISPR/Cas9 to knockout expression of the ACE2 protein in cultured lung cells, the next experiment would involve verifying the impact of ACE2 deletion on the ability of coronaviruses to infect these cells. This could be achieved by:

Exposing the ACE2-knockout cells to SARS-CoV and SARS-CoV-2 to assess whether viral entry and subsequent replication are inhibited as compared to control cells that express ACE2.

Quantifying viral replication within the cells using methods such as RT-PCR, immunofluorescence, or cytopathic effect observation.

Evaluating the cells for any unintended consequences or compensatory mechanisms that might arise due to the absence of ACE2 protein.

Through these experiments, researchers can better understand the role of ACE2 in coronavirus infection and assess whether the knockout strategy could be a viable anti-viral approach.