Modeling Gastric Cancer By CRISPR-Mediated Editing Of Tumor Suppressor Genes

Gastric cancer(GC)is one of the most common malignancies in the world.Its morbidity and mortality rank the third and fifth among all cancers.In China,the incidence of GC is the second most and only ranks behind that of lung cancer.Currently,generating a mouse model for autochthonous GC needs time with an extremely low success rate.As a powerful genome editing technology,CRISPR/Cas9 has been used to successfully develop mouse models for primary cancers,including lung cancer and liver cancer and to study initiation and development of these cancers.However,no autochthonous GC model has been reported with the CRISPR/Cas9 technology.In this thesis,I delivered a small sg RNA library established in the lab together with Cas9 into gastric cells by gastric mucosal hydrodynamic injection,inducing gastric tumors in about 7 weeks post injection.This sg RNA library targets 34 tumor suppressor genes that are important for gastric cancer.After analysis with immunohistochemistry and PCR targeted amplicon deep sequencing,I constructed the mutational atlas of targeted sites in 34 tumor suppressor genes for tumor nodules,para-tumor tissues,primary tumor cell pools and primary tumor cell clones.In these samples,many common tumor suppressor genes,e.g.p53,Arid1 a,Apc,Smad4 and Pten,have high frequencies of mutations.Based on the mutational atlas,I assembled a smaller sg RNA library including 12 tumor suppressor genes to induce gastric tumors in about three months.In these tumors,p53,Arid1 b,Smad4 and Pten were also highly mutated.In particular,with both sg RNA libraries,Arid1 b mutations existed in 2-3 types not only in tumor tissues and tumor cells but also in para-tumor tissues.This suggests that Arid1 b is a potential driver gene in gastric cancer.This work develops a protocol for development of autochthonous GC model by CRISPR/Cas9 and provides a new type of mouse model for study of gastric tumorigenesis.