Researchers prepare to screen genes associated with the replication of novel coronavirus

A study conducted at the FAPESP-funded Center for Research on Inflammatory Diseases (CRID) will identify key processes triggered by SARS-CoV-2 in human cells.

By Maria Fernanda Ziegler  |  Agência FAPESP – Researchers at the Center for Research on Inflammatory Diseases (CRID), hosted by the University of São Paulo (USP) in Brazil, are conducting research to determine which genes are key to the replication of the novel coronavirus (SARS-CoV-2) in the cells of infected individuals.

“We’re analyzing the whole human cell genome to identify the genes that play important roles in the lifecycle of the virus in infected cells. It’s a wide-ranging study, and although it won’t produce immediate answers, it will greatly increase our future understanding of the role of each gene in the biology of the virus,” CRID researcher Thiago Mattar Cunha told Agência FAPESP.

Mattar Cunha is the leader of the study, which is supported by FAPESP. CRID is one of the Research, Innovation and Dissemination Centers (RIDCs) funded by FAPESP and is based at the USP Ribeirão Preto Medical School (FMRP-USP).

Viruses use host cell mechanisms to multiply, and SARS-CoV-2 is no exception; for example, SARS-CoV-2 lacks an enzymatic apparatus owing to its simple structure and has no energy-generating system and limited resources. When a person is infected, the virus enters the human’s cells and uses the cellular machinery to make copies of its own genetic material (RNA in the case of SARS-CoV-2). A great number of replicated viruses then break through the cell membrane and proceed to infect more cells, triggering the inflammatory process typical of COVID-19.

The CRID study will investigate the ways in which host cells can facilitate or hinder viral replication. “We’re using a novel approach. The usual approach is to choose a protein or gene that appears to play a key role and investigate the actions of the virus on that basis. Our approach is to trace the influence on the viral lifecycle of the proteins encoded by the most important genes in the human genome,” Mattar Cunha said.

This approach is best used to identify genes and proteins that have not been extensively studied and helps obtain a deeper understanding of their interactions with the virus, he added.

The researchers will select the most important genes in an existing CRISPR-ko library by locating critical factors for viral infection and replication in the human genome. The CRISPR-ko library of interest has almost 80,000 guides targeting approximately 18,000 genes. The library was built using CRISPR/Cas9, a molecular gene editing tool that inserts (or deletes) nucleotides – building blocks of RNA and DNA – and even entire genes into (from) the genome. Each of the 80,000 guides corresponds to a specific location in the genome that the enzyme Cas9 can target for cleavage and subsequent editing of the genetic material.

“Each edit resembles a mutation in the region and generates what’s known as a premature stop codon. This prevents transcription of a specific gene, inactivating it and consequently stopping the cell from producing a certain protein,” Mattar Cunha said.

The “ko” suffix in CRISPR-ko refers to the “knocking out” or the deletion of specific genes, he explained. “We expect to identify between 40 and 100 genes that are associated in some way with the infection and replication process,” he said.

The next step will consist of infecting lung epithelial cells containing the gene to be activated or inactivated with a strain of SARS-CoV-2 and monitoring the role of the gene in viral replication. “After these two steps, we will have probably established a set of genes with key roles in the virus lifecycle. We’ll also know which proteins are involved in the different stages of the cycle, such as the release of messenger RNA from the virus into the cell or the development of a new capsid that will enable it to rupture the cell membrane and reach other cells,” Mattar Cunha said.

In parallel with the identification of genes, researchers are developing two strains of genetically modified (GM) mice to be infected by SARS-CoV-2. Wild-type (non-GM) mice are not susceptible to the virus.

“We’re developing these strains in the laboratory for use in various research projects here at CRID,” Mattar Cunha said. “After screening the genome and performing the experiments with cultured cells, we plan to continue the research in an animal model.”