Creative Biogene provides additional rounds of transformation and selection until every targeted gene is knocked out. Mammalian cells exposed to two or three gRNAs in turn resulted in the generation of cell lines harboring double or triple gene knockouts, that is, the successful disruption of two or three genes. All multiplexed knockout events were obtained with DNA mutations in targeted genes, and harbored the expected knockout phenotype(s). Investigation of entire gene families or biological pathways is enabled by our dual/triple gene knock out cell line generation service.