Precision Genomic Engineering of the Green Alga Chlamydomonas reinhardtii - PROJECT SUMMARY/ABSTRACT
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.
