ObjectiveTo construct and identify monoclonal Plasmodium yoelii 17XNL PALM gene knockout strain based on CRISPR/Cas9 technique.

MethodsAccording to the sequence information of P.yoelii 17XNL PALM gene in PlasmoDB database, primers and sgRNA were designed, and the left and right homologous arms were amplified by PCR.The homologous arms and sgRNA were ligated with vector pYCm-golden-blue to construct CRISPR tool plasmid pYCm-PALM KO for gene knockout.pYCm-PALM KO plasmid was transfected into P.yoelii 17XNL by electroporation.Twenty-four hours after the transfected parasites were injected into the mice via tail vein, the blood of mice was collected for examination of Plasmodium.The Plasmodium-positive mice were fed 6 mg/mL pyrimethamine to screen PALM gene knockout malaria parasites.After that, the blood was collected from the mice every 2 days and smears were taken for microscopic examination of malaria parasites.On the 8th day of screening, PCR and sequencing were performed to identify whether PALM gene knockout was successful.The red blood cells infected with PALM gene knockout malaria parasites were injected into the mice by limited dilution method.Eight days later, the blood of mice were collected to detect Plasmodium with microscopic examination and identify by PCR to screen monoclonal malaria parasite strains.The obtained PALM gene knockout monoclonal strain and P.yoelii 17XNL were inoculated into 6 Kunming mice, each inoculated with 1×10⁵ Plasmodium-infected red blood cells (iRBC).The infected mice were collected every two days to make blood smears, and the protozoa rate was calculated by Giemsa staining posterior microscopy, the data were recorded, and the SPSS 19.0 software was used for statistical analysis.

ResultsPlasmodium electrotransfected with this plasmid was screened by drug stress in mice for 8 days.The 600bp left and right homologous arms of PALM and sgRNA sequence amplified by PCR were ligated with the vector pYCm-golden-blue to obtain the CRISPR tool plasmid pYCm-PALM KO for PALM gene knockout, which was then successfully transfected into P.yoelii.The Plasmodium transfected with the plasmid pYCm-PALM KO was screened by drug pressure in mice.On the 8th day of screening, The PALM gene was detected by PCR with the internal primer of the PALM gene, and the positive recombination band was detected by PCR with the genome-specific primer, and the fragment length was 1 246 bp.The positive recombination bands were subsequently excised and sequenced, The sequencing results further confirmed that the PALM gene was knocked out successfully.On the basis of obtaining the PALM knockout strain of P.yoelii, 12 mice were infected by limiting dilution, and a single PALM knockout band was amplified from the blood of two of the mice which were used for monoclonal screening of PALM gene knockout Plasmodium parasites by PCR, and the wild-type Plasmodium could not be detected.2 monoclonal strains with 100% PALM gene knockout were obtained.The parasitemia increased with time in the two groups of mice infected with PALM gene knockout monoclonal strain and P.yoelii 17XNL wild-type strain respectively.On day 10 post infection, there was no significant differences in parasitemia levels between the two groups (P>0.05).

ConclusionsP.yoelii PALM gene knockout strain was successfully constructed and monocloned, providing preparation for the development of live attenuated pre-erythrocyte malaria vaccine.