Dataset Information

A streamlined CRISPR/Cas9 approach for fast genome editing in Toxoplasma gondii and Besnoitia besnoiti.

ABSTRACT: Toxoplasma gondii (T. gondii) and Besnoitia besnoiti (B. besnoiti) are closely related coccidian parasites belonging to the phylum Apicomplexa, which comprises many other important pathogens of humans and livestock. T. gondii is considered a model organism for studying the cell biology of Apicomplexa mainly due to the ease of propagation in diverse host cells and the availability of a wide range of genetic tools. Conversely, B. besnoiti in vitro culture systems currently exist only for the acute phase of infection, and genetic manipulation has proven much more challenging. In recent years, the targeted editing of chromosomal DNA by the programmable CRISPR-associated (Cas)9 enzyme has greatly improved the scope and accuracy of genetic manipulation in T. gondii and related parasites but is still lagging in B. besnoiti. The CRISPR/Cas9 technology enables the introduction of single point and insertion/deletion mutations, precise integration of in-frame epitope tags, and deletions of genes at reduced time and cost compared to previous methods. Current protocols for CRISPR-mediated genome editing in T. gondii rely on either constitutive or transient expression of Cas9 as well as target specific sgRNAs encoded separately or together on transfected plasmid vectors. Constitutively expressed Cas9 carries the risk of toxicity, whilst the transient approach is laborious and error-prone. Here we present a protocol for plasmid vector-independent genome-editing using chemically synthesized and modified sgRNAs. This protocol allows for rapid and cost-effective generation of mutant cell lines of T. gondii and B. besnoiti.

SUBMITTER: Winiger RR

PROVIDER: S-EPMC7865079 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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A streamlined CRISPR/Cas9 approach for fast genome editing in Toxoplasma gondii and Besnoitia besnoiti.

Winiger Rahel R RR Hehl Adrian B AB

Journal of biological methods 20201219 4

Toxoplasma gondii (T. gondii) and Besnoitia besnoiti (B. besnoiti) are closely related coccidian parasites belonging to the phylum Apicomplexa, which comprises many other important pathogens of humans and livestock. T. gondii is considered a model organism for studying the cell biology of Apicomplexa mainly due to the ease of propagation in diverse host cells and the availability of a wide range of genetic tools. Conversely, B. besnoiti in vitro culture ...[more]

PMID: 33564692

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Project description:BACKGROUND:Knowledge about parasitic infections is crucial information for animal health, particularly of free-ranging species that might come into contact with livestock and humans. METHODS:We investigated the seroprevalence of three tissue-cyst-forming apicomplexan parasites (Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti) in 506 individuals of 12 wildlife species in Namibia using in-house enzyme linked immunosorbent assays (indirect ELISAs applying purified antigens) for screening and immunoblots as confirmatory tests. We included six species of the suborder Feliformia, four species of the suborder Caniformia and two species of the suborder Ruminantia. For the two species for which we had most samples and life-history information, i.e. cheetahs (Acinonyx jubatus, n = 250) and leopards (Panthera pardus, n = 58), we investigated T. gondii seroprevalence in relation to age class, sex, sociality (solitary, mother-offspring group, independent sibling group, coalition group) and site (natural habitat vs farmland). RESULTS:All but one carnivore species (bat-eared fox Otocyon megalotis, n = 4) were seropositive to T. gondii, with a seroprevalence ranging from 52.4% (131/250) in cheetahs to 93.2% (55/59) in African lions (Panthera leo). We also detected antibodies to T. gondii in 10.0% (2/20) of blue wildebeest (Connochaetes taurinus). Adult cheetahs and leopards were more likely to be seropositive to T. gondii than subadult conspecifics, whereas seroprevalence did not vary with sex, sociality and site. Furthermore, we measured antibodies to N. caninum in 15.4% (2/13) of brown hyenas (Hyaena brunnea) and 2.6% (1/39) of black-backed jackals (Canis mesomelas). Antibodies to B. besnoiti were detected in 3.4% (2/59) of African lions and 20.0% (4/20) of blue wildebeest. CONCLUSIONS:Our results demonstrate that Namibian wildlife species were exposed to apicomplexan parasites at different prevalences, depending on parasite and host species. In addition to serological work, molecular work is also needed to better understand the sylvatic cycle and the clear role of wildlife in the epidemiology of these parasites in southern Africa.

Project description:UnlabelledToxoplasma gondii has become a model for studying the phylum Apicomplexa, in part due to the availability of excellent genetic tools. Although reverse genetic tools are available in a few widely utilized laboratory strains, they rely on special genetic backgrounds that are not easily implemented in natural isolates. Recent progress in modifying CRISPR (clustered regularly interspaced short palindromic repeats), a system of DNA recognition used as a defense mechanism in bacteria and archaea, has led to extremely efficient gene disruption in a variety of organisms. Here we utilized a CRISPR/CAS9-based system with single guide RNAs to disrupt genes in T. gondii. CRISPR/CAS9 provided an extremely efficient system for targeted gene disruption and for site-specific insertion of selectable markers through homologous recombination. CRISPR/CAS9 also facilitated site-specific insertion in the absence of homology, thus increasing the utility of this approach over existing technology. We then tested whether CRISPR/CAS9 would enable efficient transformation of a natural isolate. Using CRISPR/CAS9, we were able to rapidly generate both rop18 knockouts and complemented lines in the type I GT1 strain, which has been used for forward genetic crosses but which remains refractory to reverse genetic approaches. Assessment of their phenotypes in vivo revealed that ROP18 contributed a greater proportion to acute pathogenesis in GT1 than in the laboratory type I RH strain. Thus, CRISPR/CAS9 extends reverse genetic techniques to diverse isolates of T. gondii, allowing exploration of a much wider spectrum of biological diversity.ImportanceGenetic approaches have proven very powerful for studying the biology of organisms, including microbes. However, ease of genetic manipulation varies widely among isolates, with common lab isolates often being the most amenable to such approaches. Unfortunately, such common lab isolates have also been passaged frequently in vitro and have thus lost many of the attributes of wild isolates, often affecting important traits, like virulence. On the other hand, wild isolates are often not amenable to standard genetic approaches, thus limiting inquiry about the genetic basis of biological diversity. Here we imported a new genetic system based on CRISPR/CAS9, which allows high efficiency of targeted gene disruption in natural isolates of T. gondii. This advance promises to bring the power of genetics to bear on the broad diversity of T. gondii strains that have been described recently.

Dataset Information

A streamlined CRISPR/Cas9 approach for fast genome editing in Toxoplasma gondii and Besnoitia besnoiti.

Publications

A streamlined CRISPR/Cas9 approach for fast genome editing in <i>Toxoplasma gondii</i> and <i>Besnoitia besnoiti</i>.

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