Discovery of Zika Virus Dependency and Restriction Factors Using Flow-Based Arrayed CRISPR Screening for Identification of Targets (FACS-IT)
| Autor: | William M, McDougall, Manish, Kandpal, Jill M, Perreira, Abraham L, Brass |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Virus Cultivation
Virulence Factors Zika Virus Infection Cell Culture Techniques Zika Virus Viral Load Flow Cytometry High-Throughput Screening Assays Gene Knockout Techniques HEK293 Cells Phenotype A549 Cells CRISPR-Associated Protein 9 Humans Clustered Regularly Interspaced Short Palindromic Repeats Genetic Testing RNA Guide Kinetoplastida |
| Zdroj: | Methods in molecular biology (Clifton, N.J.). 2142 |
| ISSN: | 1940-6029 |
| Popis: | The discovery and implementation of CRISPR/Cas9 tools in pooled genetic screens have allowed for the rapid, high-fidelity identification of host-virus interactions. However, pooled CRISPR screening has significant limitations in its ability both to perform cell biology and plate reader-based screens and to find alleles that result in intermediate-strength phenotypes. Here we introduce an arrayed CRISPR screening method, FACS-IT, which allows researchers to use high content imaging analysis, plate reader assays, cell supernatant characterization, and percent infectivity to characterize CRISPR-mediated gene disruptions causing both moderate and extreme phenotypic changes. By using flow sorting capabilities and CRISPR libraries that are widely available, FACS-IT overcomes both the significant limitation of pooled screening approaches and the prohibitive costs of large-scale arrayed CRISPR reagents. In doing so, FACS-IT will enable researchers to creatively use CRISPR screening to obtain a deeper understanding of biology across a wide range of fields and applications. |
| Databáze: | OpenAIRE |
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