The eukaryotic green alga Chlamydomonas reinhardtii is a model organism for the investigation of basic questions in cell biology, cell motility and photosynthesis. In particular, studies of flagellar/ciliary assembly in Chlamydomonas have yielded key insights into the understanding of human diseases known as ciliopathies. Chlamydomonas also is being developed as a next- generation platform for carbon capture as well as the production of biofuels and biopharmaceuticals. Easily grown in quantity on defined media, Chlamydomonas is well-suited to biochemical and classical genetic analyses; collections of characterized mutants are available and methods for transformation are well established. However, work in this cell has been limited by the lack of an efficient method for targeted genomic editing. CRISPR-Cas9 approaches have found only limited success in Chlamydomonas, in part because of difficulties related to the expression of foreign gene constructs in this alga. Recently, we found that short, phosphorothioate-stabilized oligonucleotides alone can direct site-specific modification of genomic targets in Chlamydomonas. In this proposal we present our initial findings and outline specific aims to (1) optimize the use of Oligo Directed Mutagenesis (ODM) in Chlamydomonas using selectable targets, such as correction of auxotrophic markers; (2) apply ODM in a broader way to generate targeted gene knock-outs; (3) adapt ODM to introduce site-directed mutations in vivo; (4) explore the use of ODM to engineer larger genomic deletions or insertions, such as sequences encoding epitope tags; and (5) work to understand the molecular mechanisms underlying ODM in this model organism. Collectively, our research will provide powerful, new molecular tools that will be extremely valuable to the community of researchers working with Chlamydomonas and facilitate study of human disease models in this tractable organism. This project will be carried out at Haverford College (Haverford, PA), a selective liberal arts college with a strong tradition of engaging talented undergraduate students from diverse backgrounds and all walks of life and training them to be leaders in medicine, teaching and research. In addition to supporting the work of 4-5 Senior thesis students per academic year, summer stipends will be used to offer additional research experiences to sophomores and juniors from groups traditionally under-represented in science. In our prior experience, such opportunities play a pivotal role in the recruitment and retention of students in STEM disciplines.
This project develops the use of Oligo-Directed Mutagenesis (ODM) for precision genomic engineering of the green alga Chlamydomonas, a model eukaryotic organism particularly well- suited for investigation of basic cellular processes like photosynthesis as well as for understanding human diseases such as ciliopathies. We have found that phosphorothioate- stabilized oligonucleotides, introduced into cells via electroporation, can direct precision genomic editing to repair defective genes. We will develop this approach to permit gene inactivation, site-directed mutation and other modifications of the genome directly in living cells providing a novel, cost-effective and powerful approach to gene modification and analysis in a tractable model organism that can be used to investigate a number of important human conditions such as sterility, retinal degeneration, and polycystic kidney disease.
