Applying genome editing to modify the Akirin gene in the fruit fly for research
Disciplines
Biology | Cell and Developmental Biology | Molecular Genetics
Abstract (300 words maximum)
The highly conserved gene Akirin is present in flies and humans and regulates a variety of processes, from immune responses to muscle development. However, the regulatory mechanisms governing its expression and function remain largely unexplored. To better understand Akirin regulation, we first assessed its expression in the head, thorax, and abdomen of a Drosophila fly using quantitative RT-PCR. We found that this gene is expressed throughout the entire fly, but at different levels. The highest Akirin expression was observed in the abdomen, and the lowest in the thorax. For further analysis, we decided to use CRISPR/Cas9 technique to modify the endogenous Akirin gene to incorporate a small peptide (FLAG) in its coding sequence and make it trackable by FLAG-specific antibody and fluorescence microscopy. We have created a guide RNA and recombination template containing the desired sequence modification. The CRISPR efficiency was controlled by inducing modification of the gene ebony, which was irrelevant to our research but produced a tractable phenotype (i.e., black body). After screening genomic DNA from >60 individual flies, we found one hit, but it did not produce the desired modification upon further validation by immunostaining. This work is in progress, but, if successful, it will establish a new research tool to study the Akirin gene. As a future direction, we plan to employ FLAG-tagged Akirin protein in a pull-down assay followed by mass spectrometry to identify protein partners interacting with Akirin, providing deeper insight into its role in muscle formation.
Academic department under which the project should be listed
CSM - Molecular and Cellular Biology
Primary Investigator (PI) Name
Dr. Anton Bryantsev
Applying genome editing to modify the Akirin gene in the fruit fly for research
The highly conserved gene Akirin is present in flies and humans and regulates a variety of processes, from immune responses to muscle development. However, the regulatory mechanisms governing its expression and function remain largely unexplored. To better understand Akirin regulation, we first assessed its expression in the head, thorax, and abdomen of a Drosophila fly using quantitative RT-PCR. We found that this gene is expressed throughout the entire fly, but at different levels. The highest Akirin expression was observed in the abdomen, and the lowest in the thorax. For further analysis, we decided to use CRISPR/Cas9 technique to modify the endogenous Akirin gene to incorporate a small peptide (FLAG) in its coding sequence and make it trackable by FLAG-specific antibody and fluorescence microscopy. We have created a guide RNA and recombination template containing the desired sequence modification. The CRISPR efficiency was controlled by inducing modification of the gene ebony, which was irrelevant to our research but produced a tractable phenotype (i.e., black body). After screening genomic DNA from >60 individual flies, we found one hit, but it did not produce the desired modification upon further validation by immunostaining. This work is in progress, but, if successful, it will establish a new research tool to study the Akirin gene. As a future direction, we plan to employ FLAG-tagged Akirin protein in a pull-down assay followed by mass spectrometry to identify protein partners interacting with Akirin, providing deeper insight into its role in muscle formation.