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Journal of Undergraduate Research

Keywords

generating conditional cell lines, cellular and molecular research

College

Life Sciences

Department

Physiology and Developmental Biology

Abstract

Conditional cell lines are widely used throughout biochemical research. The basic concept is that you can delete a gene you are studying upon the addition of a drug, to cells in culture, and then use the knockout cells to study the genes effects. This process has been facilitated by the advent of the CRISPR/Cas9 system that allows a broad range of genome editing in living cells. Cas9 introduces a double strand break in DNA at a site specified by the CRISPR (guide RNA), and by changing the CRISPR sequence researchers can introduce these double strand breaks almost wherever they want. This in turn allows them to take advantage of a specific repair mechanism, called Homologous Recombination) to make changes to the DNA. The problem is that this repair pathway is rare and often outcompeted by the less reliable mutation prone Nonhomologous end joining (NHEJ). By creating a new construct, we can insert modified genes into the genome in an already reliable method, called Flipping In, that will allow us to then take advantage of cells’ more common repair method, NHEJ, to delete the endogenous version of our gene of interest. Finally, because of the construct we created we can delete our modified gene upon the addition of a drug. In this method we create a reliable method to create conditional genes that does not rely on a rare repair pathway.

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