Genome-Scale CRISPR/Cas9 Screening For Proliferation And Radiation-Related Genes In HCT 116 Cells

Objective:Gene editing technology provides powerful technical support for gene function study and gene therapy.CRISPR/Cas9 is an emerging genetic editing technology with higher editing capabilities.In 2013,Zhang Feng's research team firstly used CRISPR/Cas9 technology for genetic modification in mammalian cells.The sgRNA library,which greatly improved the efficiency of gene editing and reduced off-target effects,was used to knock out multiple genes.At present,the large-scale screening of CRISPR/Cas9 libraries has been applied to various fields,and including drug target genes,drug resistance genes and tumor proliferation genes screening.In this study,HCT 116 cell proliferation related genes were screened in a genome-wide manner by large-scale infection of GeCKO lentivirus libraries.On the other hand,after GeCKO lentivirus library large-scale infection,the HCT 116 cells were irradiated with?ray to screen radiation resistance related genes.Methods:Proliferation-related gene screening in HCT 116 cell:1)3 10~8 HCT 116cells were seeded and infected with GeCKO lentivirus library at 0.5 MOI for 24h.The cells were selected with 2 g/l puromycin for 6 days and 16days respectively.2)The genomic DNA of each sample was extracted,the sgRNA was amplified by nested PCR,cloned to the T vector and sequenced.3)High-throughput sequencing of sgRNA from each sapmple was performed by using the Illumina sequencing platform,and the MAGeCK software was used to analyze the genes related to cell proliferation.Radiation-related gene screening in HCT 116 cell:1)3 10~8 HCT 116 cells were seeded infected with GeCKO lentivirus library at 0.5 MOI for 24h.The cells were treated with 2 g/l puromycin for 6 days and the irradiated with 6 Gy cobalt 60.2)The cells were further cultured for 13 days and 20 days respectively.3)High-throughput sequencing of sgRNA from each sample was performed by using the Illumina sequencing platform,and the MAGeCK software was used to analyze the radiation resistance related gene.Results:1)3299 positive genes such as has-mir-8086,FOXD4L4,ACTR1B,and2919 negative genes such as POC1B-GALNT4,PLGLB2,and has-mir-2054 were obtained in cell proliferation related genes screening;472 negative screening genes with a statistically significant with a scoring threshold greater than 0.5(log2>0.5)were analyzed for Gene Ontology Analysis using DAVID Bioinformatics Resources 6.8(https://david.ncifcrf.gov).These genes were found to be mainly enriched in basic biological processes such as RNA metabolism and translation,indicating that they are essential for cell proliferation.At the same time,in GO enrichment analysis of 387positively screened genes with a score threshold greater than 0.5(log2>0.5),it was found that the main enrichment is in complement triggering,inflammatory response regulation,natural killer cell chemotaxis,etc.These genes are beneficial to Cell proliferation;2)Target gene prediction was successfully performed on the top ten miRNAs obtained from positive and negative screening.3)In the radiation resistance related genes experiments,4680 positive genes such as PHYKPL,HAUS1,FGD3,and1511 negative genes such as ZIM2,PCDHGB7 and PLN were screened.These genes are involved in multiple signaling pathways.Conclusion:1)The large-scale infection of CRISPR/Cas9 whole genome knockout library in HCT 116 cells was successfully achieved;2)The radiation resistance related genes were screened,which provided a technology for radiation resistance research.