The CRISPR-Combo method promotes genome editing in plants

Ten years ago, a new technology called CRISPR-CAS9 made it possible for scientists to change the genetic code of living things. Despite being revolutionary, the tool had its limitations. Like the first mobile phones that could perform a single function, the original CRISPR method can perform a function: removing or replacing genes in a genetic sequence.

Later iterations of CRISPR were developed for another function, allowing scientists to change the expression of genes by turning them on or off without removing them from the genome. But each of these functions could only be done independently in plants.

Now, scientists at the University of Maryland University of Agriculture and Natural Resources have developed CRISPR-Combo, a method for editing multiple genes in plants while simultaneously altering the expression of other genes. This new tool will allow combinations of genetic engineering to work together to increase functionality and improve the reproduction of new crops.

“The possibilities are truly limitless in terms of features that can be combined,” said Yiping Qi, an associate professor in the Department of Plant Science and Landscape Architecture and co-author of the research. “But what’s really exciting is that CRISPR-Combo introduces a level of sophistication in genetic engineering into plants we haven’t had before.”

The new study appears in the May 2022 issue of the journal Natural Plants.

At the same time the benefits of manipulating more than one gene outweigh the benefits of any manipulation on its own. For example, imagine the noise that is shaking through the wheat fields, threatening the livelihood and food security of farmers. If scientists removed a gene from wheat that could be affected by the disease and at the same time turned on genes that shortened the life cycle of the plant and increased seed production, they could quickly produce October-resistant wheat before they became overly susceptible to disease. damage.

That’s the kind of engineering that Qi and his team have demonstrated in four different phases of experimentation.

Step One: Prove the concept

Qi and his team developed new CRISPR methods to regulate gene expression in plants and to edit multiple genes at the same time. But in order to develop CRISPR-Combo, it was necessary to establish that these two functions of genetic engineering could be performed in parallel without negative consequences. In this new article, they have demonstrated the use of tomato and rice cells.

“As proof of the concept, we showed that we could exclude gene A and regulate or activate gene B well, without accidentally crossing gene B or regulating gene A above,” Qi said.

Qi and his colleagues then tested CRISPR-Combo on a flowering plant called rockcress (Arabidopsis), such as corn and wheat as a model for basic crops often used by researchers. Researchers have edited a gene that makes the plant more resistant to herbicides, while activating a gene that causes early flowering, which produces seeds faster. The result was a herbicide-resistant rock plant that spent eight generations a year, more than the usual four.

More efficient engineering

For the third experiment, the team demonstrated how CRISPR-Combo can improve the efficiency of plant growth by using tissue cultures from poplar trees. Reproductive programs for the development of new plant varieties generally use tissue cultures rather than seeds; consider how a plant grows its roots and leaves from a single stem planted in the ground. Scientists genetically alter the stem cells of plant tissues that have the ability to grow in whole plants, and when these plants mature and produce seeds, the seeds will undergo genetic changes.

Some plants are better at regenerating from tissue cultures than others, which makes this step the biggest blow to crop genetic engineering. The success rate of some plants is only 1%.

Qi and his team tackled the bottleneck by first editing some of the characteristics of poplar cells, then activating three genes that promote plant tissue regeneration.

“We showed in the poplars that our new method could provide a solution to the bottleneck of tissue regeneration, significantly increasing the efficiency of genetic engineering,” Qi said.

Short cut without hormones

Today, the growth of genetically engineered plants from tissue cultures requires the addition of growth hormones, which activate genes to promote growth. The research team reversed this process of rice by activating these genes directly with CRISPR-Combo. The result was rice edited by tissue culture genes that did not require hormone supplements. Qi and his colleagues found that tissue cultures grown with their method represented more of the genes edited using hormones than tissues grown using hormones.

“This method results in a very efficient genome editing process,” Qik said.

Left: Poplar buds with gene edited by CRSPR. On the right, poppy shoots with the same edition of CRSPR and gene enhancements to enhance growth. Credit: UMD.

The group has demonstrated that its CRISPR-Combo method works on a variety of plants for a variety of purposes perform experiments to test its viability in citrus fruits, carrots and potatoes in a fruit, vegetable and basic crop. They are also working to create a gold ri that is herbicide resistantce has improved nutritional content and increased red rice antioxidants.

Reference: Pan C, Li G, Malzahn AA, et al. Encourage plant genome editing with a versatile CRISPR-Combo system. Natural Plants. 2022; 8 (5): 513-525. doi: 10.1038 / s41477-022-01151-9.

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