UC San Diego

Characterizing the function of a single gene can be achieved by investigating how the gene and its protein product act in a cell or by observing how a cell responds when the gene is removed or knocked out. The ladder, called reverse functional genetics, has been greatly bolstered by the development and implementation of genetic engineering tools. Most recently, CRISPR-Cas9 genetic engineering has enabled functional genetics in a wide variety of organisms due to its ease of use and superior efficiency in gene mutagenesis compared to older technologies. Functional genetics studies in marine diatoms, photoautotrophic eukaryotic algae, have too been aided by the development of CRISPR-Cas9 genetic engineering. This study optimized the CRISPR-Cas9 tool in the pennate diatom Phaeodactylum tricornutum for bacterial-mediated conjugation cell transformation methodology. Conjugation in diatoms occurs by the exchange of an artificial chromosome or episome from E. coli to Phaeodactylum. Here, a cloning scheme was developed to permit the construction of CRISPR-Cas9 episomes that can target multiple genomic loci simultaneously. Nitrogen metabolism of marine diatoms contained unique features that gives them a competitive advantage in oceanic food webs. One feature is the compartmentalization of two glutamine-glutamate metabolic cycles (GS-GOGAT) within the chloroplast and mitochondria, respectively. This study utilized the CRISPR-Cas9 episome tool to functionally analyze a key GS-GOGAT enzyme, glutamine synthetase 2 (GS2), in Phaeodactylum by producing mutant cell lines devoid of GS2. It was found that while cell growth was not compromised, GS2 mutants accumulated lipids, a precursor to biodiesel. Lastly, the CRISPR-Cas9 episome was used to investigate this phenomenon of lipid accumulation in a nitrogen-gene mutant. To do so, a knockout library of nitrogen metabolism genes was produced. Also, the library episomes were fashioned with a novel lipid body fluorescent protein reporter that, by microscopy and flow cytometry based detection methods, was indicative of lipid production in Phaeodactylum mutants. Here, nitrogen limitation and lipid biosynthesis was uncoupled in a Phaeodactylum GS2 mutant. The expansion to high-throughput mutagenesis of a nitrogen gene library will support research into diatom biofuels.