Screening Of Promoters For SgRNA Transcription In Giardia Duodenalis And Establishment Of Transfection System

Giardia duodenalis is an important zoonotic protozoan that is parasitic in th e small intestine of the host,causing giardiasis which is distributed worldwide.The main symptom of the infected human is diarrhea.G.duodenalis has a wide range of hosts.G.duodenalis belongs to the single-cell eukaryote,whose structure is simple,the gene content is small and compact,and the coding region is extremely short.The life history of G.duodenalis is simple: the cysts which can resist in the external environment for a long time and the trophozoites which can replicate and multicply in host intetine and cause disease.Humans can become affected with G.duodenalis via contaminated food and water.G.duodenalis often causes diarrhea,nausea,vomiting and weight loss in the host,and can also cause irritable bowel syndrome and food allergies,arthritis or chronic fatigue syndrome and other complications.Nutritional status,immune status,mixed infections,and differences in normal intestinal flora of the hosts can affect the ability of G.duodenalis to infect.G.duodenalis belongs to the branch of eukaryotes and locates at the bottom of the evolution of eukaryotes.It is an important biological model for studying the evolution of higher eukaryotes.In recent years,the introduction of editable nucleases has greatly improved the efficiency of genome editing.Before the development of the technology,researchers used different CRISPR tools to destroy the genomic double-stranded DNA,thereby generating genomic variation.CRISPR/Cas9 is a typical type II CRISPR system originally discovered in bacteria and archaea.There are two key building cores: Cas9 protein and sg R NA.The sg RNA is composed of tracr RNA and cr RNA binding complex and is intracellular.Dicer III hydrolyzes its spacer and part of the repeat to form a mature sg RNA.In the last step of the interference,the sg RNA directs the Cas9 protein to a target sequence that is complementary to the sg RNA and then degrades it.In the construction of the CRISPR/Cas9 gene editing system of cells,it is necessary to cons truct a plasmid of sg RNA and Cas9 protein,insert a drug resistance gene expression cassette into the plasmid,transfer the plasmid to the cell by means of transfection technology,screen the stable expression cell line using the drug,and then conduct genetic editing studies.This editing method is simpler and less time-consuming than traditional methods and is widely used in various researches.In this study,sg RNA was used as a research object to construct a transient and stable transfection vector of G.duodenalis.The experimental analysis of each element in the knockout vector of the Trypanosome and Toxoplasma gondii was carried out,and the G.duodenalis U6 promoter was predicted,and two exogenous promoters of Trypanosoma U6 and Toxoplasma gondii 18 S r RNA and G.duodenalis U6 promoter serves as preselected promoters to initiate transcription of sg RNA.Three sg RNA expression cassettes were inserted into p Bluescript II SK(+).After successful construction and identification,the three plasmids were pur ified by electroporation.The plasmid was transfected into G.duodenalis cells and cultured for 24 h,then,the c DNA was reverse transcribed,and the promoters and abilities of the three promoters were analyzed by q PCR.After successful identification of the plasmid,q PCR was performed to detect the expression time of transiently transfected sg RNA at different time.To test whether the 3' UTR has an effect on the transcription of sg RNA,we inserted the 3' UTR of U6 into the 3' end of the sg RNA gene to construct the p BSK-GU5-sg RNA-GU3.After electroporation of G.duodenalis with two plasmids,the expression levels of sg RNA of the two vectors were analyzed by q PCR.Then,the plasmid was inserted into a NEO expression cassette to construct stable transfection vector,and G.duodenalis was cultured by G418 concentration gradient to determine the minimum G418 of lethal dose to G.duodenalis.The stable transfection plasmid was linearized with endonuclease and purified,then electrotransfected into G.duodenalis.The sg RNA stably expressed strain was screened in the medium containing G418,Finally,the genomic DNA was extracted,and NEO and sg RNA genes were detected by PCR.Through the above studies,we successfully constructed a transient transfection vector of sg RNA,and detected the sg RNA successfully transcribed by the G.duodenalis U6 promoter by q PCR.The transient expression time was 6 days,and a stable transfection vector was constructed by inserting the NEO expression cassette.Through the screening of G418 after transfection,we successfully obtained anti-G418 strains stably expressing sg RNA,and identified sg RNA and NEO genes by PCR,indicating that the stable transfection experiment was successful.This study also demonstrated by experiments that the 3 ' UTR of G.duodenalis U6 had no significant effect on the transcriptional expression of sg RNA.In this study,transient transfection and stable transfection were realized in G.duodenalis by screening and use of the endogenous promoter of G.duodenalis,which should be useful in establishment of the CRISPR/Cas9 system in G.duodenalis.