Since the agricultural revolution, mankind has strived to enhance plant varieties through genetic diversity. However, until recently, our understanding was limited to the functions of individual genes, which account for just 20% of the genome. The remaining 80%, comprised of genes grouped in families, remained a mystery on a large genomic scale.
In a groundbreaking achievement, Tel Aviv University researchers have harnessed the power of CRISPR technology to develop an innovative and scalable genetic modification method. This breakthrough allows us to uncover the roles and characteristics of duplicated genes in plants. As a result, the team has successfully identified numerous overlooked features, paving the way for a revolutionary approach to crop improvement. This remarkable development has the potential to revolutionize agricultural practices across a wide range of crops and traits, including increased yields and enhanced resistance to drought and pests.
Overcoming Genetic Redundancy
This groundbreaking research was led by postdoctoral student Dr. Yangjie Hu under the guidance of Prof. Eilon Shani and Prof. Itay Mayrose from the School of Plant Sciences and Food Security at TAU's The George S. Wise Faculty of Life Sciences. Collaborating with scientists from France, Denmark, and Switzerland, the team utilized the CRISPR gene editing technology along with bioinformatics and molecular genetics methods to develop this novel gene-location method. The research was published in the prestigious journal Nature Plants.
"We wanted to apply this technique to improve the control of creating mutations in plants for the purposes of agricultural improvement and specifically to overcome the common limitation posed by genetic redundancy," said Prof. Itay Mayrose.
Genetic redundancy, caused by gene families, has long posed a challenge in plant research. Previous methods of genetic intervention were limited by the inability to precisely identify genes responsible for specific traits. The accepted method to address this challenge is to produce mutations, that is, to modify genes in different ways, and then to examine changes in the plant's traits as a result of the mutation in the DNA and to learn from this about the function of the gene.
Thus, for example, if a plant with sweeter fruit develops, it can be concluded that the altered gene determines the sweetness of the fruit. This strategy has been used for decades and has been very successful, but it also has a fundamental problem: an average plant such as tomato or rice has about 30,000 genes, but about 80% of them do not work alone but are grouped in families of similar genes. Therefore, if a single gene from a certain gene family is mutated, there is a high probability that another gene from the same family (actually a copy very similar to the mutated gene) will mask the phenotypes in place of the mutated gene. Due to this phenomenon, called genetic redundancy, it is difficult to create a change in the plant itself and to determine the function of the gene and its link to a specific trait.
The team addressed this challenge by using CRISPR and designing sgRNA sequences that guide an enzyme called Cas9, found naturally in bacteria, to cut specific genetic sequences associated with entire gene families. Prof. Mayrose explains that "this genetic editing method allows us to design different sgRNA sequences to allow Cas9 to cut almost any gene that we want to change. We wanted to apply this technique to improve the control of creating mutations in plants for the purposes of agricultural improvement and specifically to overcome the common limitation posed by genetic redundancy."
In the first stage, a bioinformatics study was carried out on a computer, which, unlike most studies in the field, initially covered the entire genome. The researchers chose to focus on the Arabidopsis plant, which is used as a model in many studies and has about 30,000 genes. First, they identified and isolated about 8,000 individual genes, which have no family members and, therefore, no copies in the genome. The remaining 22,000 genes were divided into families, and for each family, appropriate sgRNA sequences were computationally designed. Each sgRNA sequence was designed to guide the Cas9 cutting enzyme to a specific genetic sequence that characterizes the entire family, with the aim of creating mutations in all family members so that these genes can no longer overlap each other. In this way, a library was built that totaled approximately 59,000 sgRNA sequences, where each sgRNA by itself can simultaneously modify 2-10 genes at once from each gene family, thereby effectively neutralizing the phenomenon of genetic redundancy.
In addition, the sgRNA sequences were divided into ten sub-libraries of approximately 6,000 sgRNA sequences each, according to the presumed role of the genes – such as coding for enzymes, receptors, transcription factors, etc. According to the researchers, establishing the libraries allowed them to focus and optimize the search for genes responsible for desired traits, a search that until now has been largely random.
"We believe that this is the future of agriculture: controlled and targeted crop improvement on a large scale. Today, we are applying the method we developed to rice and tomato plants with great success, and we intend to apply it to other crops as well," said Prof. Eilon Shani.
In the next step, the researchers moved from the computer to the laboratory. Here they generated all 59,000 sgRNA sequences designed by the computational method and engineered them into new plasmid libraries (i.e., circular DNA segments) in combination with the cutting enzyme. The researchers then generated thousands of new plants containing the libraries - where each plant was implanted with a single sgRNA sequence directed against a specific gene family.
The researchers observed the traits that were manifested in the plants following the genome modifications, and when an interesting phenotype was observed in a particular plant, it was easy to know which genes were responsible for the change based on the sgRNA sequence that was inserted into it. Also, through DNA sequencing of the identified genes, it was possible to determine the nature of the mutation that caused the change and its contribution to the plant's new properties.
In this way, many new traits were mapped that until now were blocked due to genetic redundancy. Specifically, the researchers identified specific proteins that comprise a mechanism related to the transport of the hormone cytokinin, which is essential for optimal plant development.
Commercialization and Future Impact
Prof. Shani concludes: "The new method we developed is expected to be of great help to basic research in understanding processes in plants, but beyond that, it has enormous significance for agriculture: it makes it possible to efficiently and accurately reveal the pool of genes responsible for traits we seek to improve - such as resistance to drought, pests, and diseases, or increasing yields. We believe that this is the future of agriculture: controlled and targeted crop improvement on a large scale. Today we are applying the method we developed to rice and tomato plants with great success, and we intend to apply it to other crops as well."
Recognizing the transformative potential of this breakthrough, Tel Aviv University's technology commercialization company, Ramot, partnered with the AgChimedes group to establish DisTree, a company dedicated to applying this technology to different crops. This collaboration, along with financial investment and professional support, aims to revolutionize agricultural genetics and ensure nutritional security in the face of climate crises.