The Development Of CRISPR/Cas9 Technique In Plasmodium Yoelii And Systematic Functional Analysis Of PyApiAP2 Transcription Factors

Malaria,caused by infection of Plasmodium parasites,remains a world-wide public health burden.Although the genomes of many malaria parasites have been sequenced,we still do not know the functions of many genes in the genomes.Studying gene function has become the focus of many studies,however,editing genes in malaria parasite genomes is still inefficient.The CRISPR/Cas9?clustered regularly interspaced short palindromic repeats and Cas9 endonuclease-mediated genome editing?system is a new powerful technique for genome editing and has been widely employed to study gene function in various organisms.In this study,we developed single gene and multiple genes editing system by using the CRISPR/Cas9 technique in the Plasmodium yoelii.Firstly,we established a single-vector pYC system that Cas9 is able to introduce site-specific DNA double-strand breaks in the P.yoelii genome which can be repaired through homologous recombination.By supplying engineered homologous repair templates,we generated targeted deletion,reporter knock-in,and nucleotide replacement in multiple parasite genes,achieving up to 100%efficiency in gene deletion?Pysera1,Pysera2,PyPDH/E1??and 22 to 45%efficiencies in knock-in and allelic replacement.The pYC system modified parasites cannot be used in second-round gene editing because of plasmid residual.In practice,it is often desired to modify multiple genetic loci in a parasite genome.On the basis of pYC system,we developed a pYC modified?pYCm?single-vector system.Drug resistant genes encoding human dihydrofolate reductase?hdhfr?and a yeast bifunctional protein?yfcu?,with cytosine deaminase?CD?and uridyl phosphoribosyl transferase?UPRT?activities in the plasmid,allowed sequential positive?pyrimethamine,Pyr?and negative?5-fluorocytosine,5FC?selections and generation of transgenic parasites free of the episomal plasmid after genetic modification.Using this system,we were able to efficiently tag a gene of interest?Pyp28?and subsequently disrupted two genes?Pyctrp and Pycdpk3?that are individually critical for ookinete motility.The CRISPR/Cas9 technique for editing the malaria parasite genome will greatly facilitate our ability to elucidate gene function.Malaria parasites have a complex life cycle with multiple developmental stages in mosquito and vertebrate host,and different developmental stages express unique sets of genes.Unexpectedly,many transcription factors?TFs?commonly found in eukaryotic organisms are absent in malaria parasites;instead,a family of genes encoding proteins similar to the plant Apetala2?ApiAP2?transcription factors is expanded in the parasites.Several malaria ApiAP2 genes have been shown to play a critical role in parasite development;however,the functions of the majority of the ApiAP2 genes remain to be elucidated.Our study systematically investigated the functional roles of PyApiAP2 genes in parasite development.Twenty-four of the 26 PyApiAP2 genes were selected for disruption,and 12 were successfully knocked out using the CRISPR-Cas9 method,pYC plasmid or pYCm plasmid.The effects of gene knockout?KO?on parasite development in mouse and mosquito stages were evaluated.Ten of 12 successfully disrupted genes,including two genes that have not been functionally characterized in any Plasmodium species previously,were shown to be critical for P.yoelii development of sexual and mosquito stages.The two new genes are Pyap2-g3?PY17X₁417400?and Pyap2-o5?PY17X₁317000?.Additionally,seven of the genes were labeled for protein expression analysis,revealing important information supporting their functions.We tagged PyAP2-G3 with 6×HA at the N-terminal end and detected protein expression in the cytoplasm and to a lesser degree in the nuclei of asexual stages and gametocytes?all the blood stages?;we also tagged PyAP2-05 with the sequence encoding 6×HA and observed protein expression using IFA.PyAP2-05 protein was expressed mainly in the nucleus and weakly in the cytoplasm of some young schizonts and strongly expressed in the nuclei of male and female gametocytes,female gametes/zygotes,and retort ookinetes but absent?or undetectable?in mature ookinetes.In addition,there were two PyApiAP2 genes?PY17X₀523100 and PY17X₁323500?that were disrupted successfully,but the parasites without the genes showed no detectable developmental defects or phenotypes in either asexual or sexual stages.The ortholog of PY17X 1323500 was disrupted successfully in P.berghei,but not that of PY17X₀523100.Our study represents the first systematic functional characterization of the P.yoelii ApiAP2 gene family and discovers important insights on the roles of the ApiAP2 genes in sex stage and mosquito transmission.