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USING A CRISPR/CAS9 KNOCKOUT TO EVALUATE THE ROLE OF FURIN IN THE INTOXICATION PATHWAY OF PSEUDOMONAS EXOTOXIN A
Author(s) -
Sanford Jack,
Zhu Yuyi,
Weldon John
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.775.21
Subject(s) - furin , crispr , pseudomonas exotoxin , proprotein convertase , microbiology and biotechnology , hek 293 cells , biology , endoplasmic reticulum , immunotoxin , cas9 , recombinant dna , cell culture , genetics , gene , biochemistry , cytotoxicity , ldl receptor , in vitro , enzyme , lipoprotein , cholesterol
Derivatives of Pseudomonas exotoxin A (PE) known as recombinant immunotoxins (RITs) are chimeric proteins being investigated as potential cancer therapies to target and kill cancer cells. RITs gain access to target cells via receptor mediated endocytosis, and traffic through the trans‐Golgi network to the endoplasmic reticulum. From there, RITs gain access to the cytoplasm where they inhibit translation elongation factor 2 (EF2) and halt protein synthesis. The subtilisin/kexin‐like proprotein convertase furin (PCSK3) has been implicated in the PE trafficking pathway as a proteolytic activator of the toxin, but circumstantial evidence suggests that furin may assist intracellular trafficking or have a combination of both cleavage and trafficking functions. This project aims to use the CRISPR/Cas9 methodology to create a furin‐deficient cell line in order to explore the role of furin in the intoxication pathway of RITs. Thus far, a targeting guide RNA has been used to direct the Cas9 endonuclease to the furin gene in HEK293 cells. The resulting cells were clonally expanded and evaluated for knockout mutations resulting from repair of the Cas9 cleavage events. Several HEK293 subclones without furin expression were identified. Initial RIT‐based cytotoxicity assays have shown a difference in susceptibility between the furin knockout and wild type HEK293 cell lines. We plan to introduce furin variants with altered function into the furin‐deficient cell line to quantifiably characterize the role of furin in the RIT intoxication pathway. Support or Funding Information I would like to thank the Fisher College of Science and Mathematics (FCSM) as well as the Towson Office of Undergraduate Research (TOUR) for their financial support of this project. I would also like to thank all of the members of Dr. Weldon's lab for their help with this research.