Discovering the Function and Characteristics of Duplicated Genes in Plants through a Wide-scale Genetic Modification Approach.

For the first time, researchers from Tel Aviv University have developed a genome-scale technology that makes it possible to reveal the roles of genes and traits in plants previously hidden by functional duplication.

The researchers point out that since the agricultural revolution, man has improved plant varieties for agricultural purposes by creating genetic diversity. But until this latest development, it had only been possible to examine the functions of single genes, which make up only 20% of the genome. For the remaining 80% of the genome, made up of genes grouped into families, there has been no effective method, on an entire genome scale, to determine their role in plant.

As a result of this unique development, the team of researchers was able to isolate and identify dozens of new features that had been overlooked until now. The development is expected to revolutionize the way agricultural yields are improved as it can be applied to most crops and agricultural traits, such as increased yield and resistance to drought or pests.

The research was conducted by postdoctoral student Dr. Scientists from France, Denmark and Switzerland also participated in the research, which was published in the journal Nature plants.

As part of the research, the team of researchers used the innovative gene-editing technology CRISPR and methods from the field of bioinformatics and molecular genetics to develop a new method for identifying genes responsible for specific traits in plants.

Professor Shani says: “For thousands of years, since the agricultural revolution, man has been improving different types of plants for cultivation by enhancing genetic diversity. But until a few years ago it was not possible to intervene genetically in a meaningful way, but only to select and enhance the desirable traits that were created The development of gene-editing technologies now allows subtle changes to be made in a large number of plants.”

The researchers explain that although gene-editing technologies, such as CRISPR, have advanced, several challenges remain that have limited their application to agriculture. One of them was the need to identify as precisely as possible the genes in a plant’s genome that are responsible for a particular trait desirable for its cultivation. The accepted way to meet this challenge is to produce mutations, that is, to modify genes in different ways, then to examine changes in plant traits as a result of mutation in the DNA, and to learn from that about the function of the gene.

Thus, for example, if you grow a plant that has sweeter fruit, it can be concluded that the altered gene determines the sweetness of the fruit. This strategy has been used for decades with success, but it has a fundamental problem: a medium-sized plant such as a tomato or rice contains about 30,000 genes, and about 80% of them do not work alone but are grouped into families of similar genes.

Therefore, if one gene of a particular gene family is mutated, there is a high probability that another gene of the same family (actually a very similar version of the mutated gene) will mask the phenotypes in place of the mutated gene. Because of this phenomenon, called genetic duplication, it is difficult to induce change in the plant itself and determine the function of a gene and its association with a particular trait.

The current study sought to find a solution to the problem of gene duplication using an innovative gene-editing method called CRISPR. “The CRISPR method is based on an enzyme called Cas9 that is naturally present in bacteria, and its role is to cut foreign DNA sequences. So the enzyme can bind to an RNA sequence, which determines which DNA sequence the enzyme needs to cut,” says Professor Myrose. These gene editing methods designed different sgRNA sequences to allow Cas9 to cut out almost any gene we wanted to alter.We wanted to apply this technique to better control mutagenicity in plants for agricultural improvement, and specifically to overcome a common limitation caused by gene duplication “.

In the first phase, a bioinformatics study was performed on a computer, which, unlike most studies in this 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 the appropriate sgRNA sequence for each family was computationally designed.

Each sgRNA sequence is designed to direct the Cas9-cutting enzyme to a specific gene sequence that characterizes the entire family, with the goal of creating mutations in all members of the family so that these genes cannot overlap with each other. In this way, a library of approximately 59,000 sgRNA sequences is built, in which each sgRNA alone is capable of simultaneously modifying two to 10 genes from each gene family, thus effectively neutralizing the phenomenon of gene redundancy.

In addition, the sgRNA sequences were divided into ten sub-libraries of approximately 6,000 sgRNA sequences each, according to the putative role of genes—such as encoding enzymes, receptors, transcription factors, etc. According to the researchers, creating the libraries allowed them to focus and improve the search for genes responsible for desirable traits, research that until now had been largely random.

In the next step, the researchers moved from the computer to the laboratory. Here, they generated all 59,000 computationally designed sgRNA sequences and engineered them into novel plasmid libraries (that is, circular DNA segments) in combination with a cutting enzyme. The researchers then generated thousands of new plants containing the libraries – each plant seeded with a single sgRNA sequence directed against a specific gene family.

The researchers observed which traits appeared in plants after genome modifications, and when an interesting phenotype was observed in a particular plant. It was easy to tell which genes were responsible for the change based on the sgRNA sequence it was inserted into.

Also, by sequencing the DNA of the identified genes, it was possible to determine the nature of the mutation that caused the change and its contribution to the new characteristics of the plant. In this way, many new traits that had hitherto been blocked due to genetic redundancy were set. Specifically, the researchers identified specific proteins that constitute a mechanism involved in transporting the hormone cytokinin, which is essential for optimal plant development.

Professor Shani says: “The new method we have developed is expected to be very useful for basic research in understanding processes in plants, but beyond that, it has great significance for agriculture: it makes it possible to efficiently and accurately reveal the set of genes responsible for the traits we seek to improve – Such as resistance to drought, pests and diseases or increased yields. We believe this is the future of agriculture: controlled and directed crop improvement on a large scale. Today we are applying the method we have developed to rice and tomato plants with great success, and we intend to apply it to other crops as well.”