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Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System
Published on: July 31, 2019
Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System
Yukie Akutsu1, Motomichi Doi1, Koji Furukawa1
1Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST).
Abstract:
Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas' disease mainly in Latin America. In order to identify a novel drug target against T. cruzi, it is important to validate the essentiality of the target gene in the mammalian stage of the parasite, the amastigote. Amastigotes of T. cruzi replicate inside the host cell; thus, it is difficult to conduct a knockout experiment without going through other developmental stages. Recently, our group reported a growth condition in which the amastigote can replicate axenically for up to 10 days without losing its amastigote-like properties. By using this temporal axenic amastigote culture, we successfully introduced gRNAs directly into the Cas9-expressing amastigote to cause gene knockouts and analyzed their phenotypes exclusively in the amastigote stage. In this report, we describe a detailed protocol to produce in vitro derived extracellular amastigotes, and to utilize the axenic culture in a CRISPR/Cas9-mediated knockout experiment. The growth phenotype of knockout amastigotes can be evaluated either by cell counts of the axenic culture, or by replication of intracellular amastigote after host cell invasion. This method bypasses the parasite stage differentiation normally involved in producing a transgenic or a knockout amastigote. Utilization of the temporal axenic amastigote culture has the potential to expand the experimental freedom of stage-specific studies in T. cruzi.
Insights
This study introduces a new method for gene knockout in Trypanosoma cruzi amastigotes, enabling essentiality studies for drug target identification. This technique facilitates research on Chagas
Area of Science:
- Parasitology
- Molecular Biology
- Drug Discovery
Background:
- Trypanosoma cruzi causes Chagas' disease, a significant health issue in Latin America.
- Validating gene essentiality in the mammalian amastigote stage is crucial for identifying new drug targets.
- Studying amastigotes is challenging due to their intracellular replication, hindering traditional knockout experiments.
Purpose of the Study:
- To develop a method for gene knockout exclusively in the T. cruzi amastigote stage.
- To enable stage-specific functional analysis of genes in T. cruzi.
- To facilitate the identification of novel drug targets against Chagas' disease.
Main Methods:
- Utilized a temporal axenic amastigote culture allowing extracellular replication.
- Introduced guide RNAs (gRNAs) directly into Cas9-expressing amastigotes for gene knockouts.
- Evaluated knockout phenotypes through axenic culture cell counts and intracellular replication assays.
Main Results:
- Successfully performed CRISPR/Cas9-mediated gene knockouts in axenic amastigotes.
- Demonstrated the ability to analyze gene function exclusively in the amastigote stage.
- Established a protocol for producing in vitro derived extracellular amastigotes for experimental use.
Conclusions:
- The temporal axenic amastigote culture system provides a powerful tool for stage-specific gene essentiality studies in T. cruzi.
- This method bypasses the need for complex stage differentiation in knockout experiments.
- The approach significantly expands experimental flexibility for T. cruzi research and drug development.
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