| CRISPR/Cpf1 is the new CRISPR system reported by Zhang Feng Group in 2015.The research proves that the system has the potential of simpler and more accurate genome editing.The helicase DDX21 can not only participate in a variety of biological processes such as messenger RNA(mRNA)precursor processing,RNA transport and degradation,ribosome production,but also in Avian influenza virus(AIV),dengue fever.Virus(Dengue virus,DENV),human immunodeficiency virus(HIV)and other viruses also play an important role in the proliferation and signal transduction process.This study firstly knocked out the DDX21 gene of mammalian HEK293 cells and infected H9N2 subtype AIV by CRISPR/Cpf1 technology,and verified the activity of CRISPR/Cpf1 system and the natural immune response mechanism of DDX21-mediated AIV infection at the cellular level;further explored CRISPR The gene editing of the/Cpf1 lentiviral system in chicken DF-1 cells and chickens laid the foundation for revealing the function of DDX21 gene and the pathogenesis of the virus.Editing mammalian cells by CRISPR/Cpf1 system:In this study,DDX21 was used as a target gene,and the target site was screened by online software chopchop to construct pU6-Lb-crRNA-DDX21 targeting vector and pcDNA3.1-hLbCpf1-RFP reporter vector,respectively.The two vectors were co-transfected into HEK293 cells and the positive cells were sorted by flow cytometry.The Cpf1nuclease cleavage activity was 56.8%by T7E1 digestion,and two DDX21 gene-stable HEK293 cell lines were obtained after screening.A new method for gene-stable gene knockout mediated by the CRISPR/Cpf1 system was successfully established.Using the constructed HEK293-DDX21-/-as the cell model,the virus titer was determined after H9N2 subtype AIV infection,and the mRNA expression level of pattern recognition receptor,cytokine and antiviral protein was detected by real-time PCR,and the knockout was further verified and revealed.Functional changes in the DDX21 gene.The results showed that the mRNA expression levels of TLR-3and MDA-5 were up-regulated,and the expression level of TLR-7 was not significantly different.The mRNA expression levels of IFN-?,IFN-?,IL-6 and OAS were down-regulated,and the difference in TCID50 was the highest.3.01 times,indicating that the DDX21 gene knockout may cause DDX21-TRIF-MyD88 signaling pathway to be blocked,inhibit the expression of type I interferon,inflammatory factors and antiviral proteins,and promote influenza virus replication.Editing chicken DF-1 cells by CRISPR/Cpf1 system:According to the design principle,four targets were screened in the second exon of chicken DDX21 gene,and the four reconstituted targeting vectors were co-transfected with DF-1 cells with the reporter vector.T7E1 digestion and TA clone sequencing showed that the target 1 cleavage efficiency was up to 33.3%(10/30),and there was a 5~48bp deletion at the target site.The Lenti-Cpf1-DDX21 lentivirus was packaged to infect DF-1 cells,and the positive cells were enriched by puromycin.The cells were sorted into 96-well plates by flow cytometry.After sequencing,two DDX21 gene knockouts were successfully obtained.DF-1 cell line.Exploration of genetically edited chicken:The lentiviral expression vector was mixed with the corresponding packaging plasmid in a certain ratio and then transfected into 293T cells,and the lentivirus titer was determined by ELISA method to be 2.5×10~7 Tu/mL;The packaged lentivirus was microinjected into the blastoderm of 527 newborn eggs,and 83 chicks were hatched,the hatching rate was 15.86%.The efficiency of lentivirus integration into the chicken genome was 22.9%(19/83).This study successfully established CRISPR/Cpf1-mediated knockout technology of HEK293 and DF-1 cells,and verified the signaling pathway involved in DDX21 by H9N2 subtype AIV infection assay,and explored the genes of CRISPR/Cpf1 lentiviral system in chickens.The editor has important application value for establishing cell and animal models and studying gene function by using CRISPR/Cpf1. |
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