Screening Genes Maintaining Mouse Embryonic Stem Cells Self-renewal Based On Genome-scale CRISPR/Cas9 Knockout Library

Embryonic stem cells(ESCs)are a group of cells derived from the inner cell mass(ICM)of the blastocyst-stage embryo and characterized by two distinctive properties: pluripotency and self-renewal.The self-renewal of ESCs refers to the ability to proliferate indefinitely in vitro and maintain three-germ layer differentiation potential under specific culture conditions.ESCs possess important research value and application prospect in regenerative medicine.In the field of ophthalmology,stem cell therapy provides hope for curing retinopathy,corneal injury and other refractory diseases by restoring normal optical function of patients.The maintenance of ESCs self-renewal is a very complex process and the underlying mechanisms have been discussed widely from endogenous transcription factors including Oct4/Sox2/Nanog pluripotent core regulatory network and MYC transcription factor network,exogenous signaling pathways including LIF-STAT3 pathway,BMP4-Smad pathway and Wnt/β-catenin pathway,epigenetic regulation like DNA methylation,histone modification and chromatin remodeling,to alternative splicing and cell-cycle machinery.Although numerous factors have been uncovered so far,there is still a lot to be discussed in the field of ESC self-renewal regulation.Exploring novel genes involved in ESCs self-renewal is a hot issue in life science research,which is of great significance for the application of stem cells.Functional genome screening is a powerful method to explore the unknown gene functions by modifying gene expression and observing corresponding cell phenotypes.With the development of technology,CRISPR/Cas9 system is a new ideal genetic screening tool.The basic principle of CRISPR/Cas9 system is to use the targeting ability of sg RNA to guide Cas9 endonuclease at specific sites in genes and introduce double strand breaks(DSBs).Based on this principle,CRISPR/Cas9 system has the advantage of high consistency and efficiency in generating targeted gene knockouts at the DNA level with high detection sensitivity and promising application prospect in high throughput screening.The CRISPR-Cas9 system has been successfully adopted in screening for essential genes,drug resistance and therapeutic targets in multiple cell lineages.In this study,we performed an unbiased genome-wide CRISPR/Cas9 screen to detect genes involved in m ESC self-renewal.We performed bioinformatics analysis based on the sequencing results.We made further selection of candidate genes for functional verification and preliminary analysis in cancer development.In conclusion,our analysis has identified genes associated with ESC self-renewal maintenance,opening up an opportunity to further understand the regulation of ESC self-renewal.The main conclusions are summarized as follows:1.Overall self-renewal screening resultsIn this study,we performed an unbiased genome-wide screen to detect genes involved in m ESC self-renewal.The sequencing data were processed by MAGe CK to score the enrichment degree of sg RNAs.Significantly varied genes were obtained by comparing Input＿d0 and total＿d14 sequencing data.Genes with the increased proportion of sg RNAs after screening(LFC>0,P < 0.05,good sg RNA≥2)were identified as UP gene group,while genes with the decreased proportion of sg RNAs after screening(LFC<0,P < 0.05,good sg RNA≥2)were identified as DOWN gene group.The results showed that UP gene group contained 1375 genes.Consistently,UP gene group contained germ layer differentiation genes and apoptosis-related genes.Cell localization analysis suggested that 54% of the total UP genes could be classified into “cellular anatomical entity”,and GO annotation showed that UP genes were mainly involved in signal transduction,cell differentiation and apoptosis in terms of biological functions.DOWN gene group consisted of 2929 genes,including essential genes for survival and important genes proved to control self-renewal.Cell localization analysis suggested that different from the UP genes,DOWN gene group was mainly distributed in the nucleus.GO annotation found that the DOWN gene group was mainly involved in transcriptional regulation,cell cycle,cell division and RNA splicing in terms of biological functions,and protein binding in terms of molecular function.KEGG pathway analysis suggested that DOWN gene group mainly focused on metabolic pathways,ribosome and spliceosome.The results of our screening included genes with known functions,but more importantly,many unknown genes,which may play an important role in the regulation of embryonic stem cell self-renewal and maintenance.2.Nuclear localization gene analysisIn this study,we focused on nuclear localization genes in DOWN group and perform further GO annotation.KEGG signal pathway analysis revealed that the DOWN group located in the nucleus mainly concentrated on splicing,ribosomal biology and RNA transport items.In order to further annotate ESC self-renewal genes and verify the screening results,Puf60,U2af2,Wdr75 and Usp16 were selected as candidate target genes through the analysis of expression profile and literature retrieval.None of these four genes has been thoroughly studied in the field of ESC self-renewal regulation.The sg RNAs for Puf60,U2af2,Wdr75 and Usp16 respectively were designed and transient knockout cells lines of them were obtained by transfection of targeted sg RNAs.Morphologic observation showed that after the knockout of target genes,the cell morphology became flat,loose and tended to differentiate.Cell counting experiments showed that the number of cells decreased after the knockout of target genes(P <0.05)with the most significant reduction in Puf60 knockout group.3.Puf60 functional analysisIn this study,we focused on analyzing the effect of Puf60 on self-renewal.EB formation experiment combined with q PCR experiment showed that the expression level of Puf60 in EB1 D and EB 3D state was lower than that in LIF/Serum state.By adding the 2i/LIF culture condition,we found that the expression level of Puf60 in 2i/LIF condition was higher than that in LIF/Serum condition.In the colony formation experiment,compared with the control group,the colony counts in Puf60 knockout m ESCs was significantly lower(P<0.001).The ratio of differentiated colonies in Puf60 knockout m ESCs was significantly higher(P<0.01)and the ratio of undifferentiated colonies in Puf60 knockout m ESCs was significantly lower(P<0.01),suggesting that the knockout of Puf60 affected the self-renewal maintenance of embryonic stem cells.There are distinct parallels between stem cells and cancer cells,like the property of self-renewal,and core members of ESC stemness genes have been proved to be involved in cancer development.Therefore,we asked the relationship between Puf60 and carcinogenesis.Compared with normal tissues,expression of Puf60 was significantly upregulated in 9different cancers: Cholangiocarcinoma(CHOL),lymphoid neoplasm diffuse large B-cell lymphoma(DLBC),glioblastoma multiforme(GBM),brain lower grade glioma(LGG),liver hepatocellular carcinoma(LIHC),pancreatic adenocarcinoma(PAAD),ovarian serous cystadenocarcinoma(OV),thymoma(THYM),and uterine carcinosarcoma(UCS).Gene chip data showed Puf60’s expression was evaluated in metastatic and tumor tissues compared with normal tissues in ovarian.Overall survival analysis was conducted to investigate the correlation between Puf60 expression and the prognosis of patients with cancers above.Results revealed that the overall survival rate of patients with low levels of Puf60 expression was significantly higher in UCS,compared with patients with high levels of Puf60 expression.TCGA database showed there was a co-occurrence trend for PUF60 and MYC in cancers.More importantly,PUF60 and MYC showed a co-expression trend in UCS.Collectively,apart from its positive role in ESCs self-renewal regulation,PUF60 as a multi-task protein is related to the development of cancers.In summary,we successfully performed a genome-wide CRISPR-Cas9 knockout virus library to screen and identify the key genes in m ESC self-renewal maintenance.Puf60,U2af2,Wdr75 and Usp16 were confirmed to be involved in self-renewal regulation.Further analysis found that Puf60 played an important role in m ESC self-renewal maintenance and was related to cancer development,which was of great significance to the better understanding of Puf60 function.As a result,our study provides a new insight into understanding ESC self-renewal regulation.