As many studies have demonstrated that mutations affecting MMR and replicative DNA polymerases can cause rapid mutagenesis and a significant increase in TMB level (28, 34, 38), therefore in our study, we generated several cell lines with different TMB levels by introducing homozygous cancer-associated MSH2 and POLE variants through the use of CRISPR/Cas9 system, and hereby developed a series of novel FFPE reference materials for TMB analysis by paraffin embedding these constructed cell lines. The gene discussed is MSH2; the disease is cancer.