Engineering And Functional Characterization Of The Small-Subunit RRNA In Plasmodium Yoelii Using CRISPR/Cas9

Malaria is a deadly infectious disease that is caused by Plasmodium spp and transmitted by female Anopheles mosquitoes.One of the remarkable features of Plasmodium parasites is the differential transcription of structurally distinct rRNA(ribosomal RNA)genes at different developmental stages.There are two types of rRNA in the genome of malaria parasites.The A-type genes(A and B units)mainly play roles in asexual stages,whereas S-type genes(C and D units)are chiefly involved in sexual or mosquito stages.Previous studies from our lab have shown that D-type small-subunit rRNA gene(D-ssu)is essential for oocyst development of Plasmodium yoelii,variations in D-ssu between P.yoelii strains and the timing of transcription could have crucial effects on oocyst development.However,the functional regulations of the individual ssu genes in parasite developmental stages are still poorly understood.rRNA molecules fold into complicated secondary structures with different domains that are critical for their functions.To study the functional roles of D-ssu ES6S and ES9S domains,we replaced D-ssu segments of P.yoelii By265 with D-ssu from P.yoelii N6 7,and exchanged D-ssu and A/B-ssu sequences in By265 using a CRISPR/Cas9 system that has been developed recently.We successfully generated several parasite clones including By265-DssuN67-Dssu,By265-DssuBy265-A/Bssu and By265-A/B ssuBy265-Dssu.Interestingly,we also obtained multiple clones of replacements such as By265-DssuBy265-A/Bssu,By265-DssuBy265-Cssu,By265-A/BssuBy265-Cssu,and By265-A/BssuBy265-Dssu without the use of homologoues DNA templates,suggesting that the homologous sequences from the endogenous ssu genes of P.yoelii can serve as templates to repair CRISPR/Cas9 generated DNA breaks.Next we infected Balb/c mice with 27 parasite clones carrying different replaced sequences from various types of ssu replacements to evaluate effects of individual replacement on the development of asexual blood stages.The majority of the parasite clones with replaced ssu segments grew similarly to the wild-type By265 parasite,except clones By265-A/BssuBy265-Cssu that had significantly lower parasitemia than those of the WT parasite on day 4-6 post-infection.Mice infected with these parasites also have higher survival rate than those infected with WT and other clonal replacement.The results suggest that the By265-A/BssuBy265-Cssu replacements may affect blood stage development of the parasite.We also fed mosquitoes with 30 individual parasite clones with different ssu replacements,counted oocysts and salivary gland sporozoites,and infected mice through mosquito bites and measured the sporozoite infectivity.Significant differences in day-8 oocyst counts were observed for most of the parasite clones compared with the WT parasite.Significant reduction in day-17 sporozoite count was also found.Additionally,clones By265-DssuR2-By265-A/B156,By265-DssuR2-By265-A/B500,By265-DssuR2-By265-C255 and By265-AssuR1-By265-D131 were not able to infect mice through mosquito bites.To confirm these finding,we constructed By265-DssR2-By265-A/B156 replacement vectors,performed additional transfections,and obtained clonal parasites having a same genotype with By265-DssuR2-By265-A/B156 parasites.However,these clones had a normal developmental phenotype in mosquito stages.These results appear to contradict the observations from those of the original parasite carrying a same replaced sequence.We are investigating the causes of the observed different results between the experiments.Off-target effects or unknown mutations could have occured during the transfection,which might cause this variation.This study efficiently produced multiple ssu replacements in P.yoelii with and without the use of exogenous templates by employing CRISPR/Cas9 system.Results from the study provide important information for studying the functions of ssu genes in malaria parasite development.