Genetic Modification Of The Protozoan Eimeria Parasite Using CRISPR/cas9 System

Eimeria parasite is one of the main pathogens that endangers the health of many animals.It is also an important model organism for studying the biological and immunological characteristics of apicomplexan parasites.Previous studies have established reverse genetic manipulation platforms such as transient transfection and selection of recombinant Eimeria.However,due to factors such as the difficulty of in vitro culture of Eimeria and low transfection efficiency,effective gene editing technology has not been established,which limits the study of Eimeria gene functions.The CRISPR/Cas9 system is an efficient and powerful gene editing tool,which has been widely used in gene editing of various organisms.In this paper,an Eimeria gene editing system based on the CRISPR/Cas9 system has been established.The major findings and conclusions are as follows:1.The E.tenella histone 4 promoter and nuclear localization sequence regulates the expression of SpCas9 endonuclease to the nucleus of the sporozoites,which is the basis for Cas9 to cut the nuclear genome of Eimeria.2.Two U6 promoters has been identified in E.tenella.Both of two promoters can effectively regulate the expression of sgRNA which with biological activity.3.The CRISPR/Cas9 system mediates the double strands breaks(DSB)of the Eimeria genome.4.The DSB can be repaired by homologous recombination in the presence of homologous fragments in Eimeria.In conclusion,we successfully developed the CRISPR/Cas9 system in Eimeria,which will accelerate gene function studies in Eimeria and apicomplexan parasites.