Periodic Reporting for period 1 - OMEGA (Developing technologies to engineer plant genomes at the megabase scale)
Reporting period: 2022-07-01 to 2024-12-31
Genomics research combined with revolutionary genome editing tools has created new opportunities to explore fundamental aspects of biology and develop novel (bio)technologies for medicine, agriculture, and industry. The OMEGA project is designed to build on these developments by enabling Megabase-scale engineering of plant genomes. Experimentally-determined lists of essential genes have been crucial for such large-scale projects in other species but are currently unavailable in plants. Therefore, the OMEGA project needs to determine which plant genes are essential for growth and development. However, the standard technologies used to identify essential genes are limited by genetic interactions (e.g. redundancy, synthetic lethality). My group is developing multiplex CRISPR screens in plants to specifically overcome these limitations. The OMEGA project will use this capability to identify genome-wide essential genes and genetic interactions in Physcomitrium patens. This will be the first systematic genetic interaction screen in plants and will likely identify interactions conserved across the green lineage. The OMEGA project will also develop technologies to move, edit, and delete DNA at the Megabase scale. As proof-of-concept, I aim to reduce ~10% of the P. patens genome and remove all non-essential DNA from one chromosome. The SCRaMbLE system will be adapted for P. patens and used as an alternative approach to minimize chromosomes and also perform in planta directed evolution. Lastly, we will use a DNA assembly method developed in my lab to build biosynthetic pathways in vivo using modular DNA assembly methods. These tools will create opportunities to explore fundamental aspects of genomics, chromosomal biology and synthetic biology. The objectives of project OMEGA are necessarily ambitious as these technologies will allow us to perform controlled, Megabase-scale engineering projects in plant genomes not possible with the current generation of genome editing tools.
We have laid the foundation to perform the genome-wide genetic interaction screen. All predicted genes have been organized into sets of 8-12 genes that we expect to interact. This is corroborated by the fact that known interacting genes are clustered together in these sets. We have also generated the necessary genotyping assays and high-throughput transfections protocols to produce the thousands of lines we will generate in project OMEGA. Work is now focused on piloting the knockout of a few hundred genes before jumping into the full screen. For the genome reduction experiments, we have been able to remove ~1 Mb of DNA from five different loci. Work is now focused on increasing the efficiency of that removal. For the SCRaMbLE system, we have demonstrated the insertion of loxPsym sites into 11 sites in the P. patens genome and have induced recombination. We are now working to increase the efficiency of delivery and scaling up the number of loxPsym sites.
For the genome-wide knockout screen we have developed protocols that will enable us to efficiently target sets of genes in a cost-effective manner. This will enable the efficient combination of any set of genes for targeting in a CRISPR experiment. We have also removed ~1% of the genomic content from P. patens. This is already a significant amount of genomic reduction for a plant species.