Validating eIF4A1 by CRISPR-Cas9 knockout as a potential target for cancer therapeutics Anna Bakewell1, Zaynab Neetoo-Isseljee1, Craig Southern1, Barbara Saxty1, Debra Taylor1 1LifeArc, Accelerator Building, SBC Campus, Stevenage, SG1 2FX, UK Eukaryotic initiation factor 4A (eIF4A) is an ATP-dependent DEAD/H box RNA helicase involved in translation initiation. Overexpression of eIF4A is observed in multiple cancer cell lines and is thought to accommodate the upregulation of translation of pro-oncogenic mRNA, thus contributing to oncogenesis. Studies have demonstrated that suppressing eIF4A1, one of the two isoforms of eIF4A that configure part of the eIF4F complex, results in reduced growth and survival of cancer cells, which is not observed when eIF4A2 is knocked down. The activity of eIF4A1 can be enhanced by pro-oncogenic transcription factors increasing translation of eIF4A1-dependent mRNAs. Many of these mRNAs encode pro-proliferative proteins involved in the cell cycle, cell survival and angiogenesis and contain highly structured 5’-UTRs containing G-quadruplexes. eIF4A unwinds these secondary structures enabling the 43S pre-initiation complex to bind to the mRNA and begin moving along it until it recognises the AUG start codon, at which point irreversible GTP hydrolysis of eIF42 prevents further unwinding and association of the 60S ribosomal subunit occurs allowing chain elongation to commence. In order to provide target validation of eIF4A1 we utilised emerging CRISPR-Cas9 gene editing technology to generate knockouts of two of the isoforms in isolation and simultaneously and developed a transient lethality assay. The gene knockouts were generated in breast cancer cell lines, MDA-MB-231 and MCF-7, using ribonucleoprotein complexes comprising Cas9 nuclease and synthetic multi-guide RNA specific to eIF4A1 and eIF4A2 genes. The results from the phenotypic assay demonstrated the effects of knocking out related genes simultaneously. Utilising CRISPR-Cas9 gene editing technology to knockout genes and develop transient lethality assays has proved to be advantageous over RNAi knockdown which we compared in- house to further validate this target.