Objective:Epidermal growth factor receptor variant III (EGFRvIII) is a constitutively-activated mutation of EGFR that contributes to the malignant progression of glioblastoma multiforme (GBM). Temozolomide (TMZ) is a standard chemotherapeutic for GBM, but TMZ treatment benefits are compromised by chemoresistance. This study aimed to elucidate the crucial mechanisms leading to EGFRvIII and TMZ resistance.Methods:CRISPR-Cas13a single-cell RNA-seq was performed to thoroughly mine EGFRvIII function in GBM. Western blot, real-time PCR, flow cytometry, and immunofluorescence were used to determine the chemoresistance role of E2F1 and RAD51-associated protein 1 (RAD51AP1).Results:Bioinformatic analysis identified E2F1 as the key transcription factor in EGFRvIII-positive living cells. Bulk RNA-seq analysis revealed that E2F1 is a crucial transcription factor under TMZ treatment. Western blot suggested enhanced expression of E2F1 in EGFRvIII-positive and TMZ-treated glioma cells. Knockdown of E2F1 increased sensitivity to TMZ. Venn diagram profiling showed that RAD51AP1 is positively correlated with E2F1, mediates TMZ resistance, and has a potential E2F1 binding site on the promoter. Knockdown of RAD51AP1 enhanced the sensitivity of TMZ; however, overexpression of RAD51AP1 was not sufficient to cause chemotherapy resistance in glioma cells. Furthermore, RAD51AP1 did not impact TMZ sensitivity in GBM cells with high O6-methylguanine-DNA methyltransferase (MGMT) expression. The level of RAD51AP1 expression correlated with the survival rate in MGMT-methylated, but not MGMT-unmethylated TMZ-treated GBM patients.Conclusions:Our results suggest that E2F1 is a key transcription factor in EGFRvIII-positive glioma cells and quickly responds to TMZ treatment. RAD51AP1 was shown to be upregulated by E2F1 for DNA double strand break repair. Targeting RAD51AP1 could facilitate achieving an ideal therapeutic effect in MGMT-methylated GBM cells.