Study On Genome Editing Of Plasmodium Berghei Using The CRISPR/Cas9 System

BACKGROUNDMalaria is an infection caused by mosquito bites,bringing malaria parasites into the blood,causing periodic regular onset of systemic chills,fever,and sweating in a short period of time.After long-term multiple episodes,it causes symptoms such as anemia and splenomegaly.Malaria are still one of the most important infectious diseases in the world.According to the latest estimates released by WHO in December 2018,there were 219 million cases of malaria worldwide in 2017,and a total of 435,000 people died of malaria infection.Especially for children under five years of age,malaria remains a huge threat to their precious lives,with an average of one child dying of malaria every two minutes.Genetic modification technology plays an important role in the biological research of Plasmodium,such as the study on function of Plasmodium gene.Due to the species specificity of Plasmodium infection,human Plasmodium can not infect model animals,so study of antimalarial drugs,drug resistance and malaria vaccines requires the establishment of a model for laboratory animals infected with human Plasmodium for in vivo studies.Although the commonly used rodent Plasmodium has a high degree of similarity in biological and drug susceptibility to human Plasmodium,such as Plasmodium falciparum,the same gene encoded by the two genomes is not identical in sequence.Moreover,some genes expressed by human Plasmodium are not present in the rodent Plasmodium genome,which makes the results of drug,inhibitor or vaccine studies using the rodent Plasmodium model unreliable.Therefore,the use of genome editing technology to transfer human Plasmodium genes into the rodent Plasmodium genome to construct animal models is of great value for Plasmodium research.Since Plasmodium is parasitic in red blood cells,the vector containing the foreign gene needs to pass through the four-layer membrane structure(host red blood cell,protozoal cell membrane,protozoal plasma membrane,protozoal nuclear membrane)to reach the cell cytoplasm of Plasmodium for recombination,which makes the transfection efficiency of Plasmodium is much lower than that of mammalian cells.In addition,due to parasitic lifestyle,about 6500 coding genes are deleted in the Plasmodium genome,and many enzymes in eukaryotic cells make deletions such as RNA interference(RNAi)technology,the genetic modification techniques commonly used in mammalian cells cannot be applied to Plasmodium.Therefore,the construction of transgenic Plasmodium requires long-term drug screening and cloning of strains,which is time-consuming,labor-intensive and inefficient,which seriously hinders the development of the research on gene function of Plasmodium.The CRISPR(clustered regular interspaced short palindromic repeat)sequence and the Cas(CRISPR-associated)protein system are naturally acquired immune systems of many bacteria and most archaea to prevent virus and plasmid invasion.The CRISPR/CAS9 system has been developed as a simple and efficient gene editing tool for gene editing research in a wide range of species.The system consists essentially of two elements: single-stranded guide RNA(sgRNA)and Cas9 nuclease,which together induce DNA double-strand breaks at specific sites on the genome.The DNA double-strand break caused by CRISPR/CAS9 system can be repaired by activating nonhomologous end joining(NHEJ)or by homologous recombination if the templates are provided.The CRISPR/CAS9 system has been shown to perform gene knock-out and knock-in in organism genes,and are significantly more efficient than other gene editing technologies.Previously,the CRISPR/CAS9 system has been applied to genetic editing of Plasmodium falciparum,Plasmodium yoelii.OBJECTIVEThis study intends to design and construct a double plasmid system based on CRISPR/CAS9 system gene editing technology to establish a genome editing technology for genome modification of Plasmodium berghei,and to select and optimize the electrotransfection protocol of P.berghei.We screen a better P.berghei electrotransfection protocol,and then use the double-plasmid genome editing technology to construct a transgenic P.berghei containing red fluorescent protein,verifying the efficiency and reliability of the double-plasmid genome editing technology.This technology makes construction of transgenic P.berghei and its in vivo model easier and more reliable and is very beneficial for future research of antimalarial drug screening,vaccine development,and gene function of Plasmodium.METHODBased on the existing plasmids pCMV/T7-NLS-T7 pol,pT71.1-ccdB,PX459,PL0035 and pmKate2-C,the P.berghei K173 strain was first cultured and passaged in Kunming mice.The whole genome DNA of P.berghei K173 was extracted,and then the basic principle of molecular biology experiments was used to construct a double plasmid system based on CRISPR/CAS9 technology,including plasmid pSGRNA-Cas9T2A-T7pol: containing T7 Pol to drive the expression of Plasmodium berghei sgRNA,Specific site targeting of the target gene of Plasmodium;pDONOR: provides a template for homologous repair at the site of targeted DNA double-strand breaks,providing a drug screening gene(hDHFR-yFcu)for screening positive recombinant Plasmodium.At the same time,the constructed intermediate plasmid PL0035-mKate2-3UTR was used to study the electroporation efficiency of Plasmodium berghei by in vitro culture of Plasmodium,purification of schizont,and flow cytometry.Plasmodium electroporation protocol.Finally,using the constructed double plasmid system modified by the double-plasmid Plasmodium berghei genome modification platform,a double plasmid which can replace the 230 p red fluorescent protein reporter gene mKate2 in the Plasmodium falciparum genome was constructed for the 230 p gene of Plasmodium berghei.In vitro culture of Plasmodium,purification of schizonts,screening of pyrimethamine,etc.,to construct a transgenic P.berghei transformed into the red fluorescent protein reporter gene mKate2 and verified by PCR amplification.RESULTSThe results of the first chapter were the successful construction of the targeting plasmid pSGRNA-Cas9T2A-T7 pol and the template plasmid pDONOR.PCR amplification,agarose gel electrophoresis,and gene sequencing confirmed that The double plasmid gene editing technology based on the CRISPR/CAS9 system was constructed successfully.The results of the second chapter of the study were obtained by screening and performing different electroporation protocols to compare transfection efficiency of Plasmodium berghei,and a better P.berghei electrotransfection protocol was obtained: Lonza Nucleofector 4D Nucleofecto System under the electroporation program FP167(P5 Primary Cell 4D X Kit),the amount of foreign DNA should be used as much as possible for transfection.The efficiency of transfection can reach 0.75%.At the same time,the growth and development of Plasmodium after electrotransfection was observed normal,and the results of transfection were identificated by PCR.The results of the third chapter are aimed at the knock-out the 230 p gene of Plasmodium berghei and knock-in red fluorescent reporter gene mKate2.The double plasmid gene editing technology was modified to obtain plasmids pSGRNA2464-Cas9T2A-T7 pol and pDONOR-230p-3UTR-5UTR.The transgenic P.berghei transformed into the red fluorescent reporter gene mKate2 was constructed and verified by PCR amplification.At the same time,it was observed that after the target plasmid with sgRNA targeting sequence was transferred into Plasmodium berghei,the growth and development of Plasmodium berghei were affected and the growth rate of parasitic rate slowed down.CONCLUSIONA genome editing technology based on CRISPR/CAS9 for P.berghei was successfully constructed,and we explored and optimized the current best electroporation protocol for P.berghei.A transgenic P.berghei that replaced the 230 p gene to a red reporter gene was constructed and the efficiency and reliability of the double-plasmid genome editing technology for Plasmodium berghei based on CRISPR/CAS9 system was verified.SIGNIFICANCE AND INNOVATIONBased on the CRISPR/CAS9 system,our study designed and constructed a highly efficient and easy-to-operate dual plasmid genome editing platform for P.berghei.We optimized the electrotransfection protocol for P.berghei to obtain a better and reliable electrotransfection protocol.The dual plasmid genome editing platform was used to construct a transgenic P.berghei containing the red fluorescent reporter gene mKate2.This study will open up the entire technical route of constructing transgenic P.berghei and,laying a foundation for the construction of subsequent transgenic P.berghei and its establishment of animal models.This study also provide a reliable and efficient technique and route of genetic modification for Plasmodium berghei on research of anti-malarial drugs,Plasmodium vaccines and studie of gene function of Plasmodium.