Evaluation of the basic functions of six calcium-dependent protein  kinases in Toxoplasma gondii using CRISPR-Cas9 system.
Toxoplasma gondii, an important protozoan parasite, infects almost all warm-blooded animals and humans. Although treatments in T. gondii are limited by the lack of effective drugs, some calcium-dependent kinases were demonstrated as the promising drug targets to chemotherapy against T. gondii due to their essential roles in T. gondii and absence from their hosts. The objectives of the present study were to investigate the functions of six calcium-dependent protein kinases (CDPK4, CDPK4A, CDPK5, CDPK6, CDPK8, and CDPK9) in T. gondii to assess whether they are suitable for designing as drug targets. We used the CRISPR-Cas9 system to disrupt six CDPK genes successfully by insertion of DHFR* at the guide RNA-targeted region in the six endogenous CDPK loci and successfully obtained the six knockout (KO)-CDPK strains. The biological characteristics of the six strains were evaluated by plaque assays, invasion, egress, replication, and virulence assays, respectively. The results indicated that there was no significant difference between the six KO-CDPK strains and wild-type strain in virulence and the lytic cycle including invasion, egress, and replication. The conclusion was the six CDPKs are not essential for T. gondii lytic cycle and also not virulence factors for mice, suggesting that the six CDPKs may participate in other functions in T. gondii.
Jin-Lei
JL
Wang
Si-Yang
SY
Huang
Ting-Ting
TT
Li
Kai
K
Chen
Hong-Rui
HR
Ning
Xing-Quan
XQ
Zhu
D000818:Animals; D064130:CRISPR-Associated Proteins / Q000235:genetics*; D064113:CRISPR-Cas Systems; D002118:Calcium / Q000378:metabolism; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D016503:Drug Delivery Systems; D005260:Female; D055786:Gene Knockout Techniques; D005838:Genotype; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D010641:Phenotype; D010957:Plasmids; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D013047:Specific Pathogen-Free Organisms; D014122:Toxoplasma / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D014774:Virulence; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D064130:CRISPR-Associated Proteins; D002118:Calcium; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases