| Babesia gibsoni,protozoans that parasitizes in the red blood cells of canines.It is mainly transmitted by Haemaphysalis Iongicornis and Rhipicephalus sanguineus.The disease causes anemia,fever,jaundice,hemoglobinuria and death in severe cases,which cause a serious threat to pet dog.Some genes play an important role in the process of invasion,replication,and escape of B.gibsoni.Editing the key functional genes of B.gibsoni can prevent host infection with parasites effectively.CRISPR/Cas9 gene editing system is the preferred tool for gene editing of Toxoplasma and Plasmodium,that has low cost,high efficiency and a wide range of applications.The CRISPR/Cas9 system cleave the genome by Cas9 nuclease with the guide of guide RNA(g RNA)to form a DNA double-strand break,then the cell repairs the DNA through homologous recombination or non-homologous end joining to achieve gene knockout,knock in,and single nucleotide mutation.At present,the only gene editing tool reported in B.gibsoni is homologous recombination.The efficiency of homologous recombination is low and easy to cause single-arm insertion.Therefore,this study tried to construct the CRISPR/Cas9 gene editing system of B.gibsoni to further expand the genome editing methods and seek a more efficient and widely used genome editing system for B.gibsoni.1.The test of BSD transient transfection: The CRISPR/Cas9 systems of Babesia bovis and Babesia divergens can achieve the selecting of gene mutant strains throuth transient expression of drug screening labels.But there is no relevant report in B.gibsoni.The BSD transient transfection system transfected into B.gibsoni and selected under drug for more than 30 days.The PCR identification results showed that during the cultivation of the first fifth generation parasites,the plasmids could still be detected in the parasites but lost after being cultured for more than six generations.And the highest parasitemia only reached 0.7% during the cultivation period.It indicates that the BSD transient transfection system can only achieve short term drug selecting in B.gibsoni.2.The stable transfection of SpCas9: It’s reported that Cas9 protein has a toxic effect on cells,and it is unknown whether Cas9 can be expressed in B.gibsoni.This study successfully replaced SpCas9 with ef-1α B.The expression of Cas9 protein in B.gibsoni was detected by Western blot.And indirect immunofluorescence assay(IFA)showed that Cas9 protein was located in the nucleus.3.The CRISPR/Cas9 knock out Tpx-1: B.gibsoni U6 promoter promote the expression of sg RNA,regarding Tpx-1 as the target gene.The constructed CRISPR/Cas9 knockout system was transfected into B.gibsoni that knocked out Tpx-1 successfully,and obtained a Tpx-1 knockout monoclonal strain.The growth rate of the Tpx-1 knockout strain was slower than the wild type,and its Tpx enzyme activity was significantly lower than the wild type.The frozen parasites of Tpx-1 knockout strain was difficult to recover,but there was no significant difference in the morphology of the parasites at different stages.In summary,the BSD transient transfection system can only achieve short term drug selecting in B.gibsoni,and the Cas9 protein can be expressed normally in B.gibsoni.Also the CRISPR/Cas9 system of B.gibsoni has achieved gene knockout at present,but the function of gene insertion and single nucleotide mutation needs further research. |
