A genome-scale CRISPR-Cas9 knockout screening may present novel perspectives on the currently undetermined pathway in nasopharyngeal cancer (NPC). To establish a basis for additional investigation into the functional mechanism of NPC, our goal was to perform a screening of its functional genes. Second-generation sequencing technology was employed with CRISPR-Cas9 library lentivirus screening in NPC cells to uncover functional genes, subsequently confirmed in NPC cells and patient tissues. In the King Edward Medical University, Mayo Hospital, Lahore laboratory, eleven radiosensitive and radioresistant genes were examined in 2023. Among these genes, in the radioresistant NPC cells, the expression of TOMM20, CDKN2AIP, SNX22, and SP1 was higher and that of FBLN5, FAM3C, MUS81, and DNAJC17 was considerably reduced (p < 0.05). In C666-1R, CALD1 was substantially upregulated. Additionally, we discovered that the deletion of genes facilitated the growth of NPC cells, whereas SP1 exhibited the opposite effects (p < 0.05). Using tissues from NPC patients, this outcome was confirmed. Furthermore, KEGG analysis suggested that the TGF-β signalling pathway and the Fanconi anaemia pathway may have played a role in NPC. There are nine genes under investigation. To increase the effectiveness of treatment for NPC, genome-scale CRISPR-Cas9 knockout screening for genes in NPC may offer fresh perspectives on the mechanisms.