Establishmentof CRISPR-Cas9 Based Genome-wide Screenins And The Application In The Preliminary Study Of Yellow Fever Virus-Host Interaction

Genome-wide Loss-of-Function Screening is a powerful tool for discovering important regulatory genes in specific biological functions,helping us better understand the underlying molecular mechanisms.Compared with the whole genome knock-down screening system based on RNA interference technology,CRISPR-Cas9 has the advantages of more efficient knockout efficiency and lower off-target effect and has been widely used in cell cycle research,cancer biology research and etc..CRISPR-Cas9 based genome-wide knockout screening system can also be applied to study virus-host interactions,helping us to discover more new host factors and better understand the molecular mechanisms of viral infection.In recent years,it has been used in various studies regarding Flavivirus virus-host interaction,and new important host factors have been discovered in the research of West Nile,Dengue and other viruses.Yellow fever virus(YFV)is the causative agent of yellow fever and is the prototype virus of Flavivirus.Yellow fever virus is mainly prevalent in the tropical and subtropical regions of South America and Africa.It is estimated that 80,000 to 200,000 people worldwide are infected with yellow fever virus annually,and about 30,000 to 60,000 people die from yellow fever virus infection.Due to the lack of understanding of the molecular mechanisms of YFV pathogenesis,the mortality rate is as high as 20%to 60%.Yellow fever virus-17D(YFV-17D)is a live attenuated vaccine strain of yellow fever virus,which is widely used to prevent YFV infection.The YFV-17D and Asibi strains have only 0.63%difference in nucleic acid sequence and 0.94%amino acid sequence difference.Due to its low toxicity,it can be cultured in the BSL-2 laboratory.These features make YFV-17D vaccine strain virus an ideal model for studying yellow fever virus.The aim of this study was to establish a CRISPR-Cas9 based genome-wide knockout screening system and to apply this system to the study of YFV-host interaction to discover key host factors regulating YFV infection.We used YFV-17D as a virus model and produced the intact YFV-17D vaccine strain virus with infectious ability.Subsequently,we verified that YFV-17D can complete the complete life cycle in Huh7 cells and produce infectious virus particles.Moreover,the cell strain would exhibit Cytopathic effect(CPE)and undergo apoptosis when challenged by YFV-17D,which make it an ideal cell model for the screening system.We used the CRISPR library GeCKO(Genome-scale CRISPR Knock-Out)to produce lentivirus library.By challenging the cells transduced with lentivirus library,we found a small population of cells survived from YFV-17D challenge and identified several potential targets by detecting the abundance of gRNA in these cells by performing Deep Sequencing.Among these targets,the gRNA targeting the gene ZNF629 was most significantly enrichedBy establishing two highly resistant monoclonal cell lines carrying the gRNA targeting ZNF629 gene,we found that the virus spread were significantly reduced in these two cell lines.These preliminary data suggest that ZNF629 may be an important host factor that positively regulates YFV-17D infection.In subsequent experiments,we need to verify whether ZNF629 is a bona fide factor positively regulating YFV-17D infection and illustrate the molecular mechanisms of the regulation.In summary,we have established a genome-wide knockout screening system based on CRISPR-Cas9 and applied it to the study of yellow fever virus-host intercation.In this system,we observed that a small portion of tranduced cells became resistant to YFV-17D infection.On this basis,we screened several host factors including ZNF629 that potentially regulate YFV-17D infection.We proved that the screening system established in this paper is easy and efficient in studying yellow fever virus and host interaction,and can be further applied to other fields such as cancer biology and stem cell research.