Background

Globally, there are active efforts to increase agricultural productivity and develop crops that can withstand adverse environments in response to climate change and population growth. Previously, CRISPR-Cas9 technology was mainly used to correct crop genomes. It has been effective in cutting genes or making small base changes, but it has been limited in its ability to accurately insert large genetic information of several kilobases (kb) into specific locations to significantly improve the useful traits of crops.

The traditional genetically modified organism (GMO) technology, which randomly inserts foreign genes into plants, often leads to unwanted gene disruption or uneven expression. Due to the dynamic gene repair mechanisms within plant cells, the success rate of insertion is also extremely low. There is a need for a new, precise genome engineering platform that can accurately install multiple genes or large regulatory sequences into specific regions of the plant genome, which has high agricultural value.

Key Findings

The research team, led by Professor Caixia Gao of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, designed PrimeRoot, a technology that can precisely transplant large amounts of DNA into desired sites in the plant genome. This editing tool is a plant-optimized Prime Editor (ePPE), which has been optimized to function efficiently in plant cells, and a system that fuses site-specific recombinases that insert external DNA in a precise direction. The ePPE first engraves a recombination enzyme recognition site at the target genome location, and then the recombination enzyme precisely merges external genetic material into this site without random replication.

To verify the performance of PrimeRoot, the research team targeted the genomes of rice and maize. The results showed that they were able to precisely insert large gene sequences of up to 11.1 kilobases (kb) into the Genomic Safe Harbor (GSH) of the plant genome without any missing or off-target effects. This demonstrates that it is possible to control large-scale metabolic pathways or introduce multiple external traits into the plant genome at once, beyond simple corrections at the base level.

To improve efficiency, process optimization was carried out in parallel. The research team established a sequential transformation system in which the gene editing material is injected twice at different times, rather than all at once. This new sequential method increases editing efficiency by 2 to 4 times compared to the existing method, and has laid the foundation for achieving a precise insertion efficiency of up to 8% in the genomes of rice and maize. In fact, the rice blast resistance gene pigmR and its related promoter were successfully inserted into the GSH of the rice genome, and plants with actual resistance were successfully produced.

Significance and Prospects

This research is expected to accelerate crop breeding and expand the field of plant synthetic biology. In the past, it was difficult to induce multiple genes involved in pest and disease resistance or adaptation to extreme climates at once with existing breeding techniques or early gene editing technologies. However, by introducing PrimeRoot, useful genes can be precisely placed at designated locations, thereby ensuring the safety and agricultural productivity of genome-edited crops.

However, there are also challenges to be solved before it can be applied directly to the agricultural field. An insertion efficiency of 6% to 8% is still considered low from the perspective of industrial production, which aims for large-scale production beyond the laboratory stage. It is necessary to conduct on-farm demonstration tests to determine whether the inserted genes function properly in plants grown in various climates over the long term. It is also a major prerequisite to complete a transient expression system to reduce potential side effects caused by long-term residence in cells.

This technology can be directly applied to scenarios for rapidly developing multifunctional crops that can cope with climate change. For example, a 10kb gene package containing rice blast resistance genes, drought resistance genes, and nutritional enhancement genes can be created. Then, PrimeRoot is used to insert it into a specific safe harbor in the plant genome in a single process. In the past, it took 5 to 10 years to individually cross and combine strains with each trait into a single variety, but with the new gene editing tool, it is possible to obtain seeds with multiple traits in just a few months. This opens up a practical pathway to rapidly introduce high-value crops that are resistant to pests and diseases and can withstand climate change to the market.