Genome-wide CRISPR/Cas9 Screen Of Hypoxia Tolerance Genes In Neuronal Cells

Background and aims:Hypoxic environment is one of the extreme environments on earth,including the high latitude hypoxic environment,such as the Tibetan plateau in China,the low oxygen environment in caves,and the periodic hypoxic environment faced by aquatic mammals.Long-term life under hypoxia will lead to a series of adverse reactions in physiology and pathology of the body.It will also lead to many diseases,such as pulmonary fibrosis,asthma,cardiovascular diseases.In all organs of the human body,neurons in the brain have the lowest tolerance to hypoxia,because almost all the energy of brain tissue comes from aerobic metabolism,and there is almost no anaerobic metabolism.In recent years,CRISPR/Cas9 gene editing technology has been widely used in many fields of biological process because of easy operation and high efficiency.This high-throughput tool makes it possible to study the contribution of genes to specific cell phenotypes at the whole genome level.Therefore,we want to find new hypoxic tolerance genes in nerve cells through the CRISPR/Cas9-sgRNA genome-wide library.Methods and conclusion:The CRISPR/Cas9-sgRNA mouse genome-wide lentivirus library infection system was optimized on the mouse neuroblastoma cell line N2a,and the genome-wide library was constructed to obtain stabile cell lines.After obtaining the data,bioinformatics was used to analyze the correlation between the samples,and then gene number and sgRNA number in the samples were identification.Then,comparison was made between the control group and the experimental group to obtain sgRNA abundance,and then gene ranking was conducted to obtain the screening results.Bioinformatics analysis was carried out with the second generation sequencing data and some candidate genes were identified.Some of these genes were already reported to be associated with neuroprotection and hypoxia.Therefore,it is feasible to screen CRISPR/Cas9-sgRNA genome-wide library in the mouse neuroblastoma cell line,which provides another applicable method for the study of hypoxic neuroprotection.