To evaluate impact of nuclear wild-type p53 induced phosphatase Wip1 on sensitivity of human colorectal cancer cell line DLD1 to the DNA damaging drugs: 5-fluouracil and oxaliplatin. Methods: Human colorectal cell line DLD1 with wild-type p53 status was used for all experiments. Stable cell line with overexpression of Wip1 (DLD1-Wip1ON) was derived using lentiviral transduction of DLD1 cells with viral particles containing human Wip1 cDNA under control of EF1a promoter. Wip1 overexpression was validated by immunoblotting. Wip1 gene knockout was performed using CRISPR/Cas9 approach. Briefly, plasmids encoding three gRNAs targeting 2nd, 3rd and 5th exon of Wip1 gene were co-transfected with three donor plasmids to produce the knock-in of selection cassette via homologous recombination. Proper cassette integration and disruption of Wip1 coding sequence were validated by PCR. Cellular toxicity was estimated by MTT essay, senescence – by SA-b-gal staining, cycle – by 7-AAD staining. Results: We showed that overexpression of Wip1 led to phosphatase accumulation in the nucleus and affected DNA damage response in colorectal cancer cells by decreasing the sensitivity of DLD1 cells to combination anti-cancer drugs used in clinics for the treatment of colorectal cancer, oxaliplatin and 5-fluouracil. Analysis of accumulation of senescence-associated beta-galactosidase showed that neither DLD1, nor DLD1-Wip1ON cells don not develop senescent phenotype and their resistance is not based on induction of senescence. To prove that resistance of DLD1-Wip1ON cells occur due to Wip1 overexpression, we created DLD1-Wip1KO cell line with bi-allellic knockout of Wip1 gene. The opposite effect was observed in cells with deletion of Wip1 making them more sensitive to combination of DNA damaging drugs. Conclusions: Overexpression and deletion of nuclear phosphatase Wip1 affects DNA damage response in colorectal cancer cells induced by a classical combination of chemotherapeutic drugs used for FOLFOX adjuvant therapy of colorectal cancer. Fundings: The reported study was funded by RFBR according to the research project № 17-04-01592.