Construction And Application Of Cas9 Transgenic Strain In Plasmodium Yoelii

Malaria is a vector-borne blood disease caused by an Apicomplexa species called Plasmodium.It was recognized a world-wide transfected disease along with Tuberculosis And HIV.Global healthy-care is becoming better,but there are billions of malaria transfection cases and hundreds thousands of death annually.It is an urgent and important project to get a better understanding of plasmodium and eradicate malaria eventually.So far,CRISPR/Cas9 is the most efficient modification tool in Plasmodium gene functional research.However,due to limited drug-resistant markers,researchers need to integrate all of the modification elements into one target vector,which causes the vector capacity to be too larsge.In addition,because of the parasitic property of plasmodium,the vector must pass through the four-layer membranes when we performed the electro-transfection,both of these reasons limit the efficiency of electro-transfection.In order to balance the relationship between targeting vector size and transfection efficiency,we integrated the 4.5kb Sp.Cas9 into the endogenous gene loci of seral,thus reducing the size of the target vector to half of the original.At the same time,we found that endogenous integrated expression of Sp.Cas9 could achieve gene transcription,translation and nuclear localization,and the positive rate of Sp.Cas9 cells was close to 100%,which was significantly higher than that of plasmid transient transfection(about 50%).We next selected two genes,cdpk3 and ctrp,which are necessary for ookinete motility,to knock out attempts.And PV membrane-located sepl and crystalloid-located dhhc10 were selected for gene tagging tests.The results showed that endogenous integrated expression of Sp.Cas9 could achieve different types of gene modification,and the smaller target vector(pYCs)not only solved the problem of low transfection efficiency caused by excessive vector size,but also improved the efficiency of molecular cloning.Compared with previous method,our newly obtained Cas9 stable-expression strain makes it flexible and efficient for gene editing.At the same time,we provide the potential for genome-scale knock-out screening in Plasmodium yoelii.