Wednesday, January 13, 2016

Analysis of non-canonical calcium dependent protein kinases in Toxoplasma gondii by targeted gene deletion using CRISPR/Cas9

 2016 Jan 11. pii: IAI.01173-15. [Epub ahead of print]


Calcium dependent protein kinases are expanded in apicomplexan parasites, especially in T. gondii where 14 separate genes encoding these enzymes are found. Although previous studies have shown a role for several CDPKs in controlling invasion, egress, and cell division in T. gondii, the role of most of these genes is unexplored. Here we developed a more efficient method for gene disruption using CRISRP/Cas9 that was modified to completely delete large, multi-exonic 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 function of non-canonical CPDKs. 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 lead to reduced plaquing, fitness defect in a competition assay, and reduced tissue cysts 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 function of many genes in T. gondii.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
[PubMed - as supplied by publisher]

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