Discovery Of Proteins Involved In Cellular Cholesterol Trafficking By CRISPR-Cas9 Library

Cholesterol is an important lipid for all mammalian cells,which plays an essential role in a plethora of biological processes,such as regulation of membrane integrity and fluidity,biosynthesis of bile acids and steroid hormones.It is involved in the formation of cell membranes and is a precursor to the synthesis of bile acids,sterols and some vitamins.The stability of intracellular cholesterol levels depends on cholesterol efflux,absorption and endogenous synthesis.The exogenously provided LDL enters the early endosomes through the endocytosis of LDLR,and is hydrolyzed by cholesterol esterase to become free cholesterol,which is delivered from the early endosomes sorting to the late endosomes/lysosomes.NPC1 and NPC2 play important roles in the transport of cholesterol in the lysosome,then cholesterol is transported out of the late endosome and lysosome.In this thesis,we want to know whether there are other important proteins involving cholesterol trafficking.Combining Amphotericin B treatment and Crispr-Cas9 library,we want to screen new proteins that play role in cholesterol transport.After five-time repeats,we enrich cells that show cholesterol accumulation by flow cytometry.At last,we get candidate genes by analyzing the high throughput sequencing data.Functional experiments show genes that involve in cholesterol transport.Now,we regard 67 genes as candidate genes.After classifying these candidate genes,we found that these genes are involved in intracellular vesicular transport,lipid metabolism and other pathways.We will obtain candidate genes through Amphotericin B screening,as well as RNAi and phenotypic verification of gene knockout cells,and then find some genes that may affect cellular cholesterol transport through confocal laser microscopy.The interaction proteins of these genes were found by mass spectrometry,and the function of this gene was later explored in the animal model of mice under physiological conditions.By determining the function of these genes,it was possible to find new proteins in intracellular cholesterol transport.By determining the function of these genes,we may find new proteins participating in intracellular cholesterol transport.Intracellular cholesterol transport disorders can cause serious diseases.The purpose of this study is to find new proteins that affect intracellular cholesterol transport,study the detailed mechanism of intracellular cholesterol transport,and find potential treatments for diseases caused by intracellular cholesterol transport disorders.