Study Of Regulatory Factors Of Wnt/?-catenin Signaling Using CRISPR/Cas Screen Technology

Wnt/?-catenin signaling pathway is highly conserved in evolution.During embryonic development,it plays a key role in cell differentiation,proliferation,migration and other processes.In adult tissues,it plays an important role in the maintenance of adult stem cell self-renewal ability and tissue damage repair.The dysregulation of Wnt signaling is not only critical for tumorigenesis,but also plays a key role in the metastasis of advanced tumors.Therefore,Wnt signaling pathway is a very attractive anti-tumor drug target.The main research objective of this project is to systematically search for important regulatory factors involved in Wnt signaling pathway,and to study the functional mechanism of these novel signaling molecules,and to study its significance in tumor cells.This study combines the Wnt/?-catenin signaling pathway reporting cell line and CRISPR/Cas genome-wide screening technology,through two rounds of screening and bioinformatics analysis,to identify signal molecules involved in the downstream regulation of Wnt signaling pathway.To verify the screening results,multiple screened genes were selected and knocked out one by one in the reporter cells.In the reporter cells after gene knockout,we measured the expression of the Wnt downstream gene Axin2 by RT-QPCR and the expression of ?-catenin at the protein level by WB,thereby verifying whether the screened signal molecules are indeed regulating downstream of the Wnt signaling pathway.The first round of screening enriched 460 genes,of which the known Wnt signaling molecules accounted for 3.5%,the second round enriched 29 genes,and the known Wnt signaling molecules accounted for 20.7%.This indicates that our second round of screening is more efficient in enriching genes associated with the Wnt signaling pathway.Through two rounds of library screening,a total of 9genes(APC,CSNK1A1,SKP1,CUL1,GSKIP,DKKL1,IFI44 L,KCNC2 and NOT7)negatively regulating Wnt signaling pathway were identified.Among them,APC,CSNK1A1,SKP1,CUL1,GSKIP and DKKL1 genes are known negatively regulating Wnt signaling pathway.There is no report on the other three genes(IFI44L,KCNC2 and NOT7)on the negative regulation of Wnt signaling pathway.Our results show that combination of CRISPR/Cas9-based genome-wide genetic screen and reporter gene technology can be used to identify genes involved in regulation of signaling pathways.It can be widely used to study different signaling pathways in different contexts to answer any biological questions onwhich phenotypical separation can be applied.This study identified three unreported genes associated with the Wnt signaling pathway(IFI44L,KCNC2 and NOT7).So far,these specific functional mechanism are still being studied..