Location: Foreign Animal Disease Research
Project Number: 8064-32000-063-010-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2019
End Date: Dec 31, 2022
This project seeks to generate Genome-scale CRISPR Knock-Out (GeCKO) cells to screen against African swine fever virus (ASFV) to identify host genes involved in ASFV replication and infection. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) -Cas9 technology will be used to generate the single-gene knockout cell libraries. Following selection of resistant cells by ASFV infection, the remaining viable cells are subjected to next generation sequencing in order to identify which host genes are critical for virus infection. The intended results of the proposed work is to identify host targets for a better understanding of the replication strategies of ASFV and for the development of ASFV prophylactics and vaccines.
To generate the GeCKO cells for screening, the lentiviral vector GeCKO v2 system will be used. The Cas9 nuclease, sgRNA, and antibiotic resistance gene for selection, are all incorporated into a lentivirus vector which can then be introduced to the cell line of interest. The antibiotic resistance gene is used for selection of only those cells which successfully incorporate the lentivirus. Sequencing is used to identify the gene knockouts. The generated GeCKO cell line consist of a mixture of cells carrying single-gene knockouts which can be used for screening studies using ASFV. Vero and/or COS cell lines will be used for generating the GeCKO cell libraries, and ASFV strains adapted to these cells will be used for the virus infection screening process. Once gene targets are identified, subsequent knockout studies can be performed in target host cells with sgRNAs designed specifically against the respective host gene sequence. The generated GeCKO cells will be used to screen for genes critical for ASFV infection. The surviving GeCKO cells are subsequently sequenced to identify the resulting cell genotypes, ie to identify the genes which are critical for ASFV replication. To confirm the effect of gene knockouts on ASFV replication, small/short interfering RNAs will be used to temporarily knock-down expression of the genes identified in the original genome wide analysis. Vero/COS cells are then infected and virus replication evaluated by expression of viral genes, proteins and/or infectious virus progeny. Positive target genes will be confirmed in susceptible porcine cells.