Analysis of Noncanonical Calcium-Dependent Protein Kinases in Toxoplasma gondii by Targeted Gene Deletion Using CRISPR/Cas9

Abstract

Calcium-dependent protein kinases (CDPKs) are expanded in apicomplexan parasites, especially in Toxoplasma gondii where 14 separate genes encoding these enzymes are found. Although previous studies have shown that several CDPKs play a role in controlling invasion, egress, and cell division in T. gondii, the roles of most of these genes are unexplored. Here we developed a more efficient method for gene disruption using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPRassociated protein 9) that was modified to completely delete large, multiexonic genes from the genome and to allow serial replacement by recycling of the selectable marker using Cre-loxP. Using this system, we generated a total of 24 mutants in type 1 and 2 genetic backgrounds to ascertain the functions of noncanonical CDPKs. Remarkably, although we were able to confirm the essentiality of CDPK1 and CDPK7, the majority of CDPKs had no discernible phenotype for growth in vitro or infection in the mouse model. The exception to this was CDPK6, loss of which leads to reduced plaquing, fitness defect in a competition assay, and reduced tissue cyst formation in chronically infected mice. Our findings highlight the utility of CRISPR/Cas9 for rapid serial gene deletion and also suggest that additional models are needed to reveal the functions of many genes in T. gondii.

Genetic Mapping of Pathogenesis Determinants inToxoplasma gondii

“…This break can then be repaired by nonhomologous end joining to create frame-shift or stop mutants, or by homologous recombination to create allelic replacements or knockouts when targeting constructs are employed. CRISPR has been successfully applied to all these genetic manipulation strategies in T. gondii, and in strains that were previously refractory to genetic manipulation (7,8,71,107,112). The power of this very efficient approach promises to open a myriad of possibilities for the study of this parasite.…”

Genome-Wide Identification and Evolutionary Analysis of Sarcocystis neurona Protein Kinases

“…In T. gondii , downregulation of CDPK1 interfered with parasite motility, host cell invasion and egress [43], while disruption of CDPK3 caused defective parasite egress [67]. Further, the essentiality of CDPK6 and CDPK7 in T. gondii has recently been demonstrated [68]. Indeed, TgCDPK1 has been targeted for the development of new drugs for toxoplasmosis [69].…”

New mechanisms of disease and parasite-host interactions

“…To test the first one -whether parasites with genetic disorders accumulating metabolites affect host's health -mutant parasites may be generated in the laboratory using cutting edge technology such as CRISPR (reviewed in [7]). This technique allows the generation of mutations in specific genes in a myriad of species, including parasitic protozoans such as Toxoplasma gondii [8], Cryptosporidium parvum [9] and could be applied for nematode parasites [10][11]. Therefore, it would be possible to rapidly generate parasite strains in the laboratory harboring mutations in diverse metabolic pathways.…”