Deletion of the GAS Regulatory Sequence of IFN- γ Induced Gene(ICAM-1) by Using CRISPR Cas9 to Decrease the Jak3/Stat3Dependent IDO Expression to Repress Tumor Cells Development

Abstract

IDO is a checkpoint molecule that regulates T-cell proliferation. Since IDO contributes to T cell death and creates anenvironment for cancer cells to grow, it is necessary to regulate IDO expression. Previous studies demonstrated thatIFN-gamma-induced genes would indirectly activate IDO expression through the Jak/3 pathway, and ICAM-1 is oneof the IFN-gamma-induced genes. This work investigates the effect of ICAM-1 on IDO expression and the effect of2 GAS segment knockouts on ICAM-1 expression. CRISPR-Cas9, heat shock transformation, PCR, and restrictiondigestion are used to knock out the target sequence (GAS) and insert the successful knocked-out gene into the E. coli.RT-PCR is utilized to measure the expression level of ICAM-1 and IDO after the introduction of successfully knockedout genes. The possible result of this work presents that both GAS gene knockouts are thriving; the new gene withoutGAS – 2787 bps expresses less ICAM-1 protein than the gene without GAS – 115 bps, and ICAM-1 does not affect IDOexpression at all. Results of this work indicate that the GAS sequence that is farther away from the ICAM-1 RBS sitemight decrease the ICAM-1 expression level less than does GAS sequence that is nearer to RBS, and ICAM-1 might nothave any direct impact on IDO expression, which means that tumor cell development cannot be inhibited by regulatingICAM-1 expression. Future studies could research more on functions of IFNgamma-induced genes other than ICAM-1and their relationships with IDO expression.