Gene-editing technologies have already made a big impact in medicine and biotechnology, where they are being used to modify the genes that cause disease. The utility of methods like CRISPR, TALEN, and ZFN to edit the genome of organisms continues to expand. Plants are no exception. Plants modified using gene-editing technology have no traces of foreign DNA, which thus far has meant that they are not included in regulations that cover genetically modified organisms (GMOs).

CRISPR is an acronym for clustered regularly interspaced short palindromic repeat. It’s a technology that targets a specific DNA sequence and destroys it. CRISPR is adapted from the immune systems of microorganisms. When threatened by a viral infection, the CRISPR machinery takes a DNA sequence from the invading virus and inserts it into the CRISPR sequence with saved DNA from other viruses. The sequence is then transcribed into RNA that guides the Cas9 protein to the matching viral sequence, which is then cleaved, rendering it nonfunctional. The cutting mechanism in the CRISPR-Cas9 system is provided by two small RNAs. Since the cell will attempt to repair the cleaved DNA, CRISPR can be harnessed to “edit” that DNA by including a template with the desired sequence that will hybridize to the damaged section during the repair process.

TALENs—transcription activator-like effector nucleases—are restriction enzymes that can bind and cleave specific DNA sequences. TAL effectors are derived from Xanthomonas bacteria with a conserved repeat sequence of 33–34 amino acids that bind the target DNA sequence. Zinc finger nucleases (ZFNs) are another class of DNA binding proteins that cleave DNA and can be engineered for gene editing. A ZFN has two functional domains. One domain contains “finger” modules that bind DNA. The other domain is a DNA cleaving protein.

Early successes

Gene editing has already been used to modify common crop plants like rice, wheat, barley, and potatoes. The technology is straightforward to use, and could be a way to move forward with plant breeding programs while avoiding the lengthy regulatory process for GMOs.

Gene editing has already been used to modify common crop plants like rice, wheat, barley, and potatoes…is straightforward to use, and could be a way to move forward with plant breeding programs while avoiding the lengthy regulatory process for GMOs.

“They’re looking for traits for resistance to disease, drought tolerance, or resistance to herbicides,” explains Melissa Kelley, Ph.D., a senior research and development leader for the GE Healthcare Dharmacon portfolio. GE Dharmacon offers a line of CRISPR-Cas9 products including reagents that can be used for editing plant genomes (Dharmacon Edit-R). “We’re seeing that being able to precisely change a sequence of a gene even by just a few or a handful of bases, you can either change or modulate the expression level so that you can make plant species with better nutritional value or more aesthetically pleasing traits. A popular one right now is nonbrowning mushrooms.”

Nonbrowning mushrooms created with CRISPR-Cas9 technology have created a sensation among plant researchers. Pennsylvania State University scientists engineered a white button mushroom (Agaricus bisporus) by targeting genes for polyphenol oxidase (PPO), the enzyme that causes mushrooms to brown. Deleting a small number of base pairs from one of six PPO genes resulted in a decrease of enzyme activity by 30%. In 2016, the U.S. Department of Agriculture said that it would not regulate the mushroom.1

As another example, DuPont Pioneer developed and commercialized waxy corn hybrids using CRISPR-Cas technology. Waxy corn has a high amylopectin starch content and is milled for many consumer products like processed foods, adhesives, and paper. Waxy corn traditionally has lower yields than nonwaxy varieties. The CRISPR-edited hybrids are designed for higher yields, and like the nonbrowning mushrooms, the USDA has said it would not regulate them.

David Douches, Ph.D., is program director for the Michigan State University Potato Breeding and Genetics Program. The goals of the program are to create improved varieties of potatoes with reduced use of pesticides, better nutrition, and sustainable farming practices. The potato (Solanum tuberosum) is an important food crop that can be grown commercially in any of the 50 U.S. states. Genetic improvement of the potato is complicated by an autotetraploid genome, asexual propagation, and a number of other traits of the plant. Genome editing offers an opportunity to modify traits of the potato while avoiding the pitfalls of conventional genetic engineering and plant-breeding methods.

Douches has been working with the CRISPR-Cas9 system to target the gene for acetolactate synthase1 (StALS1) using Agrobacterium tumefaciens (Agrobacterium). StALS1 has a potential role in herbicide resistance.2 His group has done similar work using TALENs. Douches says that they have had more success with TALENs because of off-targeting effects with CRISPR, but that TALENs are more labor-intensive to create. “CRISPR is a one-day process,” Douches says. “TALENs take a week.”

Challenges

Paul Dabrowski, CEO of Synthego, agrees that plant genome editing is an important and growing area of research. “With human population expected to exceed 10 billion by 2050, there is great value in increasing crop yield, seed diversity, and creating disease or pest-resistant plants. Furthermore, breeding plants that don’t elicit allergic responses is of huge interest. Drought-resistant plants, or plants tailored to their specific climate and geography will be needed to feed the world,” said Dabrowski.

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Synthego markets synthetic sgRNA that is suitable for plants and has recently introduced a guide design tool with more than 40 curated plant genomes.

According to Dabrowski, CRISPR technology makes genome engineering simpler and more precise, and thus it is easier to validate the results. This means that research and development with plants becomes more accessible, and new companies are able to launch the next generation of plant modification.

A critical difference between plants and mammalian systems is delivery of CRISPR components in plants. Plants have a tough cell wall that resists penetration by genetic machinery. Some plants can be formed into protoplasts by removing the cell wall, and transfected using lipid-based reagents. However, not all protoplasts can be induced to re-form plants. Plant embryos can be transformed with ribonucleoproteinss using a "gene gun."

“To introduce CRISPR in plants is a little more challenging than mammalian systems,” says Savita Bagga, a global marketing manager with MilliporeSigma. In addition to the tough cell walls, Bagga says that the plant life cycle can be a challenge. “You have to go through the whole phase of starting seeds before you can see results.”

Furthermore, plasmid-based CRISPR editing can't be used in plants as there is a significant risk of incorporating plant DNA into the host genome of plants. In vitro transcribed (IVT) guides have similar risks, since they are generated using DNA and could pose a problem if any DNA is retained through the IVT purification process. Synthetic RNA and purified nuclease protein for non-GMO research has emerged as the cleanest, most transient way to edit plants. “Prior to CRISPR, plant breeding might take years. With CRISPR, plant traits can be obtained within months of beginning a project,” Dabrowski adds.

Other hurdles in editing plant genomes include:

    • Slow growth of plants, prolonging the process of validating phenotypes
    • Multiple copies of chromosomes (e.g. strawberries, which have an octoploid genome) complicate the process of creating homozygous changes
    • Diversity of cell types in plants makes delivery of gene-editing components difficult
    • Plant genomes are not well annotated, and gene editing requires precise knowledge of the sequence to be changed.

Plant gene editing faces nontechnical challenges, as well. “Arguably, the biggest challenge to using CRISPR for plant-based applications is the same as applications in other organisms, and that is the restrictions and uncertainty around the intellectual property,” says Benjamin Borgo, a senior global product manager for Agilent Technologies. According to Borgo, Agilent has taken a proactive approach to licensing CRISPR, and is committed to removing as many IP barriers as possible.

Agilent recently launched SureGuide custom CRISPR libraries that provide researchers with the ability to fully design their own pooled libraries for any application, including targeting within plant genomes. These libraries can contain anywhere from 100 to 100,000 guides per library and can be used for functional genomics applications including knock-outs, knock-ins, or transcriptional control.

References

1. Waltz, E., “Gene-edited CRISPR mushroom escapes US regulation,” Nature, 532(7599), 293-293, 2016. [PMID: 27111611] 

2. Butler, N. M., Atkins, P. A., Voytas, D. F., & Douches, D. S, “Generation and Inheritance of Targeted Mutations in Potato (Solanum tuberosum L.) Using the CRISPR/Cas System,” Plos One,10(12), 2015. [PMID: 26657719] 

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