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CRISPR-Cas9 Editing in Plants: What's the Latest Research?

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The emergence of CRISPR-Cas9 genome-editing tools created new opportunities for enhancing plant and crop traits in agricultural biotechnology research. Now, multiplex genome-editing (MGE) technologies, which allow for simultaneous modification of several genetic sites in a single genome in the same experiment, are promising to further transform the field.   

Here, we discuss the latest research advancements in research applying multiplexed CRISPR-Cas9 editing in plants, including novel approaches to enhance the efficiency of large-scale genome-editing projects.

Altering gene expression in crops without introducing foreign DNA

Since the emergence of CRISPR-Cas technology as a gene-editing tool, numerous research labs have utilized it to decrease the expression of specific genes in crops.

In a new study from the Innovative Genomics Institute at the University of California Berkeley (UCB), scientists applied CRISPR-Cas9 in rice crops to reach a different end goal: increased gene expression.

“Past research has shown that this tool can be used to decrease expression of genes involved in important trade-offs, such as those between plant architecture and fruit size,” Dr. Dhruv Patel-Tupper, an AAAS Science and Technology Policy Fellow at the United States Department of Agriculture and the study’s lead author, said. “This is the first study, to our knowledge, where we asked if we can use the same approach to increase the expression of a gene and improve downstream activity in an unbiased way.”

Patel-Tupper is a former postdoctoral student in the lab of Professor Kris Niyogi at UCB, which focuses on photoprotection, methods to improve photosynthesis and carbon dioxide removal. Photoprotection refers to biochemical processes that help organisms defend against and manage damage from the sun.

The genes that encode these biochemical processes naturally occur in all plants. In a 2018 study, Niyogi and colleagues found that overexpression of Photosystem II Subunit S (PsbS), using transgenic methods, enhanced a model crop’s water-use efficiency. Inspired by this work, the new study aimed to alter the expression of a plant’s native genes without having to insert foreign DNA. The research team hypothesized that this could be achieved by applying CRISPR-Cas9 gene editing to regulatory DNA mechanisms upstream of PsbS in rice, an important crop for global food supplies.

By “flipping” the regulatory DNA, PsbS expression was successfully increased to an extent that surprised even the research team.

“The changes in the DNA that increased gene expression were much bigger than we expected and bigger than we’ve really seen reported in other similar stories,” Patel-Tupper said. “We were a little bit surprised, but I think it goes to show how much plasticity plants and crops have. They’re used to these big changes in their DNA from millions of years of evolution and thousands of years of domestication. As plant biologists, we can leverage that ‘wiggle room’ to make large changes in just a handful of years to help plants grow more efficiently or adapt to climate change.”

Increased levels of the PsbS protein enhanced a photoprotective mechanism called non-photochemical quenching and water-use efficiency.

Using RNA sequencing, the research team explored whether their strategy had affected the activity of other genes that carry important functions in the rice genome, identifying a “very small” number of differentially expressed genes.

As only one percent of the plants generated by the team possessed the desired phenotype, there’s a long road ahead for this method to be optimized, the researchers emphasized. Eventually, it could help overcome regulatory barriers associated with genetically modified and genetically edited organisms, Patel-Tupper said: “We showed a proof-of-concept here, that we can use CRISPR-Cas9 to generate variants in key crop genes and get the same leaps as we would in traditional plant breeding approaches, but on a very focused trait that we want to engineer and at a much faster timescale.”

An image of an individual in rice fields.

Rice fields. Credit: iStock.

“It’s definitely more difficult than using a transgenic plant approach, but by changing something that is already there, we may be able to preempt regulatory issues that can slow how quickly we get tools like this into the hands of farmers,” he concluded.

Boosting sugarcane yield to enhance biofuel production

Sugarcane is an economically important crop that provides ~70% of the world’s sugar and is grown across 121 different countries. Due to its high sucrose content, sugarcane is becoming increasingly important to the biofuel industry as feedstock for ethanol production.

The crop’s complex genome – it is a hybrid of Saccharum officinarum and Saccharum spontaneum – has hindered efforts to improve sugarcane via conventional breeding methods. Thankfully, sophisticated genome-editing tools, such as CRISPR-Cas technology, can lend a hand.

What are biofuels? 

Biofuels are fuels derived from biomass, such as crops and agricultural waste. They are an environmentally friendly form of energy and are emerging as an attractive alternative to fossil fuels such as coal and oil.

Researchers from the University of Florida’s Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) recently utilized CRISPR-Cas9 technology to edit the genome of sugarcane which, on the phenotype level, resulted in an adjusted leaf angle. As a result, the sugarcane leaves captured a greater amount of sunlight, ultimately increasing the biomass produced by the crop.

An image of sugarcane.

Sugarcane. Credit: iStock.

Dr. Fredy Altpeter, professor in molecular genetics, plant breeding and biotechnology at the University of Florida, led the research, published in Plant Biotechnology Journal.

“This is the first peer-reviewed publication describing a field trial of CRISPR-edited sugarcane,” Altpeter said. “And this work also shows unique opportunities for the editing of polyploid crop genomes, where researchers can fine-tune a specific trait.”

Sugarcane’s complex genome stems from the fact that it has many copies of each gene, which means that each individual plant’s phenotype is dependent on the collective expression of a specific gene. Altepeter and colleagues’ project was possible because CRISPR-Cas9 technology can be designed to edit as little or as many copies of a gene as a researcher desires.

In previous work by the same team, a gene known as LG1 was identified as important for the formation of joints between the leaf blade and sheath, causing leaves to stand upright. Sugarcane’s genome possesses 40 copies of LG1, enabling Altpeter and colleagues to mutate a varying number of copies across different plants and study the effects on biomass in field trials. “In doing so, we were able to tailor the leaf architecture until we found the optimal angle that resulted in increased biomass yield,” Altepeter said.

One line of the mutated crop – L35 – possessed edits in ~12% of the LG1 copies. In field trials, L35 demonstrated a 56% decrease in leaf inclination angle and a subsequent 18% increase in yield.

“The scalable co-editing of LG1 in highly polyploid sugarcane allows fine-tuning of leaf inclination angle, enabling the selection of the ideotype for biomass yield,” the research team said.

Enhancing the efficiency of large-scale genome editing in plants

The discussed research studies demonstrate how MGE using CRISPR-Cas technology is increasing in popularity among agricultural researchers.

Dr. Thomas Jacob’s lab at the VIB-UGent Center for Plant Systems Biology is exploring new approaches to develop easy-to-use genome editing systems for a variety of plant species, with a specific focus on developing methods that reduce the complexity and costs associated with large-scale genome-editing projects.

In the lab’s latest study, published in The Plant Journal, it has developed novel screens that systematically mutate thousands to hundreds of thousands of genes at one time in the model plant Arabidopsis thaliana (Arabidopsis).

“Here, we systematically tested different nuclear localization sites (NLS) and promoter configurations for the production of inheritable, multiplex mutants in Arabidopsis,” the authors explained.

In CRISPR-Cas9 editing, promoters are DNA sequences that regulate Cas9 expression, while NLS’ are short amino acid sequences that ensure Cas9 reaches its target site within the nucleus.

Jacobs and colleagues tested up to seven promoters and six NLSs in simplex and multiplex editing experiments. They found that a combination of using Ribosomal protein S5 A (RPS5A) promoter to express Cas9, and flanking Cas9 with bipartite NLS, resulted in the most multiplex-edited plants – 99% of plants contained at least 1 knockout mutation, while over 70% had 4–7 mutations. 

“This represents a significant advancement in the field of plant genetics and provides a reliable and efficient tool for researchers who focus on complex genetic engineering. What I find particularly interesting is the effect of the NLS. I daresay it had a stronger effect than the promoter,” Jacobs said.

The study marks the highest multiplex editing efficiency achieved in Arabidopsis to date, and the researchers are confident that the optimizations made will likely apply to other CRISPR systems.

Transforming plant biology and enhancing crop development

Knowledge obtained through MGE experiments is facilitating advancements in both basic and applied agricultural research.

As laboratories continue to refine approaches to MGE – boosting its efficiency and ability to fine-tune genetic editing of crops – these technologies carry significant potential for increasing new plant biology discoveries and enhancing our ability to generate resilient crops in the face of global crises.