Collaborative Research: EAGER: Development of an Artificial Chromosome System in Chlamydomonas Based on CENH3 Tethering

Some of the most pressing problems in agriculture and biotechnology can only be tackled by large scale changes in crop or host genomes where multiple genes must be introduced and inherited together for successful trait improvement. For example, desirable crop traits such as disease resistance, improved yields and resistance to climate change may require multiple genes introduced into one strain or cultivar, a task that is not practical with current transgenic methods.

Artificial chromosomes have the potential for solving this problem, but the technology for creating and deploying plant artificial chromosomes remains inadequate. Under this proposal a green alga and established research organism that is related to land plants, Chlamydomonas reinhardtii, will be used to accelerate the design and implementation of artificial chromosomes.

Because of its easy cultivation and rapid generation time (4 hours versus 3+ months for most plants) Chlamydomonas artificial chromosomes can be developed much faster than in land plants. The lessons learned from successful implementation of artificial chromosomes in Chlamydomonas can be used to fast-track the creation of artificial chromosomes in land plants and will have important immediate benefits by enabling the modification and improvement of algae as sources of biomass, biofuel, and high value pharmaceuticals and nutraceuticals which cannot be easily or cheaply produced using existing biotechnology.

This proposal aims to address the most significant challenge in any effort to design a functional synthetic chromosome in eukaryotes, the centromere. Centromeres in all eukaryotes are defined by the presence of the histone H3 variant CENP-A/CENH3. The Dawe laboratory has demonstrated the feasibility of activating new plant centromeres using a simple tethering approach based on the DNA binding domain of the LexA repressor to its operator, LexO, which serves as a synthetic centromere organizing center.

In this method a host strain expressing a native CENH3 sequence fused to LexA is generated. Next, an array of LexO binding sites is incorporated into a small synthetic chromosome containing telomeric sequences and selectable markers on both arms. Preliminary data from the Dawe laboratory shows that in plants the LexA-CENH3 protein binds to the LexO array and recruits additional CENH3 to create functional centromeres.

Under this proposal a similar method will be adopted for the alga Chlamydomonas as a potentially game-changing advance in synthetic biology. This proposal combines the established expertise of the Dawe laboratory in plant artificial chromosomes and the Umen laboratory in algal genetics and molecular biology to test and establish an artificial chromosome system in Chlamydomonas.

Under this proposal 1. Chlamydomonas centromeres will be fully sequenced and validated using long-read sequencing methods, and formally validated/defined using chromatin immunoprecipitation with a centromere marker protein, CenH3. 2. Transgenic Chlamydomonas strains expressing its native CenH3 paralogs as fusion proteins to LexA will be created and validated. 3.

Chlamydomonas artificial chromosomes of different sizes and configurations (e.g., linear, circular) and containing LexO arrays for centromere nucleation will be built and tested for centromere assembly, mitotic/meiotic segregation, stability, and gene expression.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.