Whole Genome CRISPR/Cas9 Lentivirus Library Amplification And Its Application In Studying Of PCSK9 Function

Lipoprotein is a complex of apolipoprotein,lipid and other small subcomponents that carries lipid in the blood.Among them,increased low density lipoprotein(LDL)level and decreased high density lipoprotein(HDL)level are crucial risk factors for atherosclerosis and coronary heart disease.The LDL receptor(LDLR)is the main receptor for LDL clearance,and plays critical roles in controlling the blood cholesterol level.Proprotein convertase subtilisin/kexin type 9(PCSK9)is a newly identified secreted protein that plays an important role in regulating blood cholesterol level.Our and others previous studies find that PCSK9 promotes the degradation of LDL receptor,thus regulates LDL-C level in the blood.Human genetic studies also shows that gain-of-function mutations of PCSK9 lead to increased level of LDL-C and increased the incidence of coronary heart disease,while its loss-of-function mutations lower blood LDL-C level and protect human from coronary heart disease.So PCSK9 has been an attractive drug target for cardiovascular disease.Although PCSK9 inhibitors have been developed as cholesterol-lowering drug,the molecular mechanism of PCSK9-mediated LDLR degradation is still largely unknown.Deep understanding of the molecular mechanism of PCSK9-mediated LDLR degradation will help us better evaluating the safety of PCSK9 inhibitors,and also provide new ways for drug development.CRISPR/Cas9 is a new generation of gene editing technology.The interval repeat sequences element and its related enzyme Cas9 found in bacteria and archaea genome can control the target DNA double-strand break.Guided by a 20bp single strand RNA,the Cas9 enzyme will cut DNA and cause double-strand break in specific position.During the repairing of DNA double-strand break,miss match or frame shift will happen and lead to inactivation of certain genes,which has become a powerful tool for studying the gene function.The genome-scale sgRNA library is also a very useful reagent for screening functional genes at the genome level.The current thesis first amplified the CRISPR/Cas9-sgRNA plasmid library and packed them into lentivirus.By deep sequencing,we found that this library has very good quality.Using this library,we screened genes involved in PCSK9 function at whole genome level.Our screening results dozens of candidate genes,which pave the way for further functional study.