(A) Western blot analysis of MTR in HCT116, 8988T and HepG2 clonally isolated lines after treatment with lentiviral CRISPR-Cas9 targeting scrambled control (control-1) or MTR (ΔMTR-1 and ΔMTR-2). Results are representative of 3 biological replicates with similar results. (B) Folate concentrations in mouse and human serum measured by LC-MS (mean ± SEM, 6 technical replicates for pooled human serum, n = 7 male CD-1 nude mice on control diet with 4mg/kg folic acid and n = 7 male CD-1 nude mice on low folate diet). (C) Growth of subcutaneous HCT116 xenografts in female CD-1 nude mice on standard chow with 3 mg/kg folic acid (mean ± SEM, n = 10 mice, P values were determined by mixed-effects two-way ANOVA comparing tumors implanted on mice’s two flanks: wild-type (WT) and ΔMTR-2, control-1 and ΔMTR-1). (D) Growth of subcutaneous HCT116 xenografts in male CD-1 nude mice on a low folate diet with or without leucovorin supplement (mean ± SEM, for low folate diet: n = 7 mice for control-1, n = 8 mice for ΔMTR+eGFP, n = 15 mice for ΔMTR+MTR; low folate diet with leucovorin supplementation: n = 8 mice for each cell line).