Mutagenesis of Ptdss1 Gene, Using CRISPR/Cas9 System in Drosophila

Abstract

Due to the increase in average life expectancy, there is growing interest in research on aging-related diseases, especially neurodegenerative diseases. Phospholipids are a major component of the cell membrane and are also highly concentrated in nerve cells. Phospholipid composition is altered in the brain in neurodegenerative disease. Previous studies have shown that phosphatidylserine synthase1 (ptdss1) mutant Drosophila has characteristics of neurodegenerative disease. However, existing strains of ptdss1 mutants are knockdown mutations created by P-element insertion at regulatory sites, which limits their use in researching the specific role of ptdss1 in neurodegeneration. To determine the more precise function of ptdss1, knockouts with mutations in the coding sequence are necessary. In this study, knockout mutations were constructed using the CRISPR/Cas9 system. For ptdss1 mutagenesis, target site gRNA expression mutations were produced and crossed with a germline-specific Cas9 expression line. In germ cells of the flies carrying both the Cas9 transgene and the gRNA transgene, a Cas9-gRNA complex was formed, making an indel mutation. These flies were crossed with wild type flies to construct heterozygous mutants. Mutant flies were characterized by T7 Endonuclease I assay and DNA sequencing. Three types of knockout mutations were constructed, reducing RNA expression levels to about 70% of wild type flies. They are expected to using model animal to configure out the function of ptdss1.