Improvements Of CRISPR/Cas9 Technology For Precise Deletion Length And Establishment Of Mouse Liver Cancer Model Base On This Technology

This thesis is consisted of two major research topics:1)Harnessing accurate non-homologous end joining for the CRISPR/Cas9 genome editing with precise deletion length;2)Establishing primary liver cancer mouse model by CRISPR/Cas9.Topic 1:Previous studies have shown that over 60%of I-SceI-induced non-homologous end joing(NHEJ)is accurate.Repair profiling of two CRISPR/Cas9-induced DSBs that are hundreds or thousands base pairs apart has also revealed that a significant portion of distal ends of these two DSBs are repaired by accurate NHEJ.To determine the extent to which proximal ends of CRISPR/Cas9-induced DSBs are rejoined by accurate NHEJ,we designed paired sgRNAs to guide Cas9-induced DSB pair that are 40-120bp apart at endogenous loci and evaluated accurate NHEJ.We found that over 50%of NHEJ is accurate in mouse and human cells.Compared to I-SceI-induced NHEJ,CRISPR/Cas9-induced NHEJ at an endogenous locus is similarly regulated by the core NHEJ factor XRCC4,providing an accurate and convenient reporter-less NHEJ assay.We also elevated paired sgRNAs-guided gene knockout(out-frame deletion)and in-frame deletion of particular gene regions and found improved gene editing.Inhibition of CtIP promotes bias toward accurate NHEJ without lowering overall NHEJ efficiency,yielding a possible strategy to further improve genome editing requiring precise deletion length.This study not only develops a convenient and reliable reporter-less NHEJ assay.But also provides a way to improve the CRISPR/Cas9 genome editing that requires precise deletion length.Topic 2:Primary liver cancer is highly malignant and associated with multiple etiologic factors,various genetic alternations and a high level of heterogeneity.Study of liver cancer genomics has already provided us a large amount of information about genetic alterations.However,it is unclear what the driver mutations or driver mutation combinations are and what the crosstalk is between mutated genes.Here we used the CRISPR/Cas9 system to edit 34 tumor suppressor genes associated with liver cancer with a sgRNA library in mice and induced formation of liver tumors within 30-60 days.These liver tumors exhibited a complicated pattern of mutations,possibly suggesting the influence of genomic instability,polyploidy in liver cells and heterogeneity of liver cancer.We are currently taking single cell analysis to characterize these liver tumors,in hope to identify key mutations and mutation combinations that drive formation of liver cancer.This study also provides a rapid and effective method for development of primary liver cancer mouse models.