Endogenous Gene Tagging of pfr2 and pfr5 in Trypanosoma cruzi using CRISPR/Cas9

Document Type

Oral Presentation

Campus where you would like to present

Ellensburg

Event Website

https://digitalcommons.cwu.edu/source

Start Date

15-5-2019

End Date

15-5-2019

Abstract

Trypanosoma cruzi, the causative agent of Chagas disease, is a single-celled parasite that contains a unique structure called the paraflagellar rod (PFR). The PFR is a lattice-like structure of cytoskeletal filaments that extends along the length of the flagellum. The exact functions and protein composition of the PFR has yet to be determined, although several major and minor proteins have been identified. The PFR is not only a complex structure shown to be crucial for cell motility, but it is also part of a group of proteins that have shown to protect mice from a lethal dose of T. cruzi following immunization. PFR5 is a proposed component of the PFR, containing a region of homology (PFR domain) which has been found within several well-characterized PFR proteins. Currently, it is unknown if PFR5 is associated with the PFR, with one of the focuses of this study being on its localization. PFR5 localization is being determined using a new gene tagging technique that utilizes CRISPR/Cas9 technology, with PFR2 localization serving as a proof of principle since its localization is well known. This technique allows for the precise insertion of a small hemagglutinin tag at the end of a gene of interest, with subsequent protein product also containing the tag. Localization of the tagged proteins can then be visualized using fluorescent antibodies. Successful utilization of this technique, as well as localization of the PFR5 protein, will help to further research and understanding of this unique structure.

Faculty Mentor(s)

Gabrielle Stryker

Department/Program

Biological Sciences

Naomi Bryant - SOURCE 2019.pptx (14499 kB)
Slides for SOURCE 2019 presentation Bryant

Additional Files

Naomi Bryant - SOURCE 2019.pptx (14499 kB)
Slides for SOURCE 2019 presentation Bryant

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Endogenous Gene Tagging of pfr2 and pfr5 in Trypanosoma cruzi using CRISPR/Cas9

Ellensburg

Trypanosoma cruzi, the causative agent of Chagas disease, is a single-celled parasite that contains a unique structure called the paraflagellar rod (PFR). The PFR is a lattice-like structure of cytoskeletal filaments that extends along the length of the flagellum. The exact functions and protein composition of the PFR has yet to be determined, although several major and minor proteins have been identified. The PFR is not only a complex structure shown to be crucial for cell motility, but it is also part of a group of proteins that have shown to protect mice from a lethal dose of T. cruzi following immunization. PFR5 is a proposed component of the PFR, containing a region of homology (PFR domain) which has been found within several well-characterized PFR proteins. Currently, it is unknown if PFR5 is associated with the PFR, with one of the focuses of this study being on its localization. PFR5 localization is being determined using a new gene tagging technique that utilizes CRISPR/Cas9 technology, with PFR2 localization serving as a proof of principle since its localization is well known. This technique allows for the precise insertion of a small hemagglutinin tag at the end of a gene of interest, with subsequent protein product also containing the tag. Localization of the tagged proteins can then be visualized using fluorescent antibodies. Successful utilization of this technique, as well as localization of the PFR5 protein, will help to further research and understanding of this unique structure.

https://digitalcommons.cwu.edu/source/2019/Oralpres/47