CRISPR Libraries and Whole-Genome Screening to Identify Essential Factors for Viral Infections.

Abstract

The CRISPR-Cas9 system has revolutionized genetics and offers a simple and inexpensive way of generating perturbation that results in gene repression, activation, or editing. The advances in this technique make possible the development of CRISPR libraries which consist of a set of sgRNAs to cause perturbations in several genes in the same cell population. The use of libraries raised the CRISPR-Cas9 technique to a genomic scale and provides a powerful approach for identifying previously unknown molecular mechanisms and pathways involved in a specific phenotype or biological process. More specifically, the CRISPRko libraries (set of sgRNAs for gene knockout) and their high-throughput screenings are widely used in research with viral agents, and it was enlarged even more with the COVID-19 pandemic. With this chapter, we aim to point out how this tool helps in understanding virus-host relationships, such as the mechanisms of virus entry into the cell, the essential factors for its replication, and the cellular pathways involved in the response against the pathogen. The chapter also provided some practical considerations for each step of an experimentation using these tools that include choosing the library and screening type, the target cell, the viral strain, the library amplification and guaranteeing its coverage, the strategies for the gene screening pipeline by bioinformatics, and finally, target validation. To conclude, it was presented a table reviewing the last updates in the research for antiviral therapies using CRISPR libraries.