Endogenous Promoter Screening And Transient Expression Of CAS9 Gene In Giardia Duodenalis Trophozoties

Giardiasis is a zoonotic protozoan disease caused by Giardia parasitizing the duodenum with diarrhea as the main symptom.Hosts infected with Giardia will have different symptoms due to variability in immunity.The hosts with normal immunity mostly have self-limited diarrhea,while the immunocompromised hosts,especially the AIDS patients,will suffer from colliquative diarrhea,which is life-threatening for severe cases.The disease is distributed worldwide and highly infectious.The host will be infected immediately after intake of only about 10 infective cysts.Therefore,Giardia has also been listed by WHO as one of the most easily overlooked pathogens.Giardia is a relatively old lower eucaryotic organism,which has a unique intracellular ultrastructure(such as the presence of peripheral vesicles and lack of mitochondria,typical Golgi apparatus and peroxidases,etc.).However,in terms of proliferation,Giardia retains the binary fission like prokaryotes and includes two relatively simple life stages(the trophozoite and the cyst),making itself an important model organism for studying the differentiation and molecular mechanism of higher eukaryotes or other parasite cells.To study the molecular mechanism of Giardia,genetic manipulation is necessary.Editing genome of Giardia will help us to understand the gene functions,clarify its pathogenic mechanism and virulence factors,and provide new drug targets for prevention and treatment of giardiasis.In recent years,the CRISPR-CAS9 gene editing system has been discovered and widely used in various fields of life sciences.In ths editing system,CAS9 protein serves as a functional core and the CRISPR fragment containing the target gene sequence works as an activator for realizing knock-out or knock-in of a target gene.As it is only necessary to construct the above two components into a plasmid and transfect it into cells,stable cell lines after gene editing can be obtained via drug selection.The tool has the advantages of low cost and simple operation compared to the traditional gene editing methods.In this study,we used Toxoplasma gondii CRISPR-CAS9 gene editing vector as an example.This vector has been successfully used in knockout of some T.gondii genes.The components of the vector were analyzed in details.With pBluescript plasmid as the vector backbone,5'-UTRs of GDH,Actin,?-giardin,?-giardin and CWP1 genes as endogenous promoters(including the promoter sequences of key genes such as CA_nT frame and A/T cassette),and the 3'-UTR(containing polyA tail)of TUB gene as transcription and splicing factors,five Giardia CAS9 transfection vectors were constructed.The vectors were transfected into Giardia trophozoites by electroporation.After cultivation for 24 hours,the trophozoites were collected and observed under a fluorescence microscope.The efficiency of Giardia endogenous promoters was assessed.Meanwhile,the expression of CAS9 protein in Giardia trophozoites was assayed by indirect immunofluorescence and western blot.The results showed that only the GDH promoter could effectively drive and express CAS9 protein.After the trophozoites were smeared and placed under a fluorescence microscope,it was found that the EGFP-CAS9 fused protein was green.Indirect immunofluorescence experiment further confirmed the expression of CAS9 protein and its wide distribution in the cytoplasm of trophozoites.Western blot results showed that the fused protein had a molecular mass of approximately 189 kDa,which is consistent with the predicted value.The Giardia CAS9 gene expression vector constructed in this study was a transient expression vector.In order to evaluate the residence time of the vector in trophozoites under the promotion of the GDH promoter,the Giardia trophozoites after electroporation were divided into groups according to the incubation time.Each group of trophozoites was collected by“ice bath method”,and the total RNA was extracted and reversely transcribed into cDNA.With cDNA as a template,qPCR was adopted to detect relative expression of CAS9 genes in different time groups.Meanwhile,with?-antin gene as an internal reference,the tendency of CAS9 gene transcription was evaluated by?CT method.The results showed that the gene transcription level of Giardia CAS9 gene peaked at 2 days after transfection and the gene could stay in the cells of trophozoites for 5 days.This study provides a theoretical basis for the expression of CAS9 protein by Giardia endogenous promoter,and has a positive significance in the establishment of a novel Giardia gene editing tool.