Mike May earned an M.S. in biological engineering from the University of Connecticut and a Ph.D. in neurobiology and behavior from Cornell University. He worked as an associate editor at American Scientist, and he is the author of hundreds of articles for clients that include Nature, Science, Scientific American and many others.
People were farming plants by at least 9,000 BCE. They probably selected the best ones soon after, and that marked the beginning of modifying crops. Centuries of improvements in selection and breeding followed. Today’s scientists leverage the rapid advances in molecular tools as a means to modify crop plants. Molecular approaches are enabling them to more accurately and efficiently target and carry out specific alterations, such as improved growth or resistance.
In the early 1990s, Francisco Mojican at the University of Alicante in Spain started working on clustered regularly interspaced short palindromic repeats, known today as CRISPR sequences. Now the CRISPR/Cas9 system is used relatively easily to modify an organism’s genome. Scientists can also use TAL effector endonucleases (TALENs) to break DNA strands and then edit them.
The keys to these technologies are ease of use and the ability to reliably modify the genomes of crop plants to make them easier to grow in difficult conditions, resist pests or provide higher yields.
Aiming accurately
Early work with TAL effectors came from Jens Boch and Ula Bonas from Martin-Luther-University Halle-Wittenberg Germany [1] and Adam J. Bogdanove of Cornell University and Daniel Voytas of the University of Minnesota in Minneapolis [2].
Today, scientists in Bogdanove’s lab use CRISPR/Cas9 and TALENs. Bogdanove says they use “CRISPR/Cas9 for its ease, and TALENs because they are also not hard to generate and we can use them at sequences that CRISPR/Cas9 is unable to target.” For example, a target needs a protospacer adjacent motif (PAM) sequence to signal Cas9 to bind and cleave the DNA appropriately.
“We are using the technology to understand gene function and to test the behavior of genetic variants in different genetic backgrounds in rice,” Bogdanove adds. For example, these scientists use gene-editing technology to test how a particular version of a gene from one variety of rice behaves in a different variety of rice. “We do not have any products in development,” Bogdanove says, “but the understanding we are gaining from our basic research will lead to crops with improved disease resistance and tolerance to abiotic stresses, such as drought.”
Analyzing the edits
At Pacific Biosciences, scientists developed Single Molecule, Real-Time (SMRT) Sequencing, which can be used to accurately measure results of gene editing. “The benefit of SMRT Sequencing for measuring the outcome of genome-editing studies has been described in a number of publications,” says Jonas Korlach, chief scientific officer of Pacific Biosciences [3, 4]. “With the new Sequel System, which provides higher throughput compared with the predecessor PacBio RS II system, genome-editing outcomes can be measured in less time and more cost-effectively.” He adds, “Barcoding and pooling multiple samples is possible through PacBio sample barcoding solutions.”
This technology can be used with the most popular nuclease platforms, including CRISPR/Cas9, TALENs and zinc finger nucleases (ZFNs). Korlach points out a variety of benefits of using this technology, including the ability to target any site and optimize gene-targeting parameters. He adds that this platform “can detect large insertion/deletion gene-editing events.” On top of all of that, he notes that the system is “highly accurate, fast and inexpensive.”
Scientists, says Korlach, can apply this technology “to crop plants in a straightforward manner.” He concludes that “training requirements are minimal, as standard PacBio sequencing is carried out, [and] the data output and analysis—alignment and mapping, variant calling—are also all standard protocols.”
Commercial and custom
Thermo Fisher Scientific develops products for gene editing and modulation and also provides custom services. These products and projects can utilize CRISPR or TALEN technologies. For example, the company has exclusively licensed TALE based gene-editing technology from the 2Blades Foundation, which retained rights for engineering to battle plant diseases.
“One of the main challenges with plants is delivering the gene-editing tools to the cells,” says Jon Chesnut, senior director of R&D at Thermo Fisher Scientific. “We have some collaborations ongoing where we are trying to create editing systems that are efficient enough to solve this and hopefully to increase the throughput.” That would allow plant scientists to develop screening technologies in which they try a range of edits to a plant’s genome, pick the ones that look the most promising and then grow them to see how they really work. “This would reduce testing time,” Chesnut says.
With today’s technology, it remains a challenge to pick out the plant cells that have been edited as intended.
With delivery remaining an issue in plants, says Chesnut, “the downstream piece of selecting the edited cells can be like looking for that needle in a haystack.” Still, he adds, “CRISPR and TALENs really improve the efficiency, which used to be really terrible with other techniques.”
Still, this market segment often needs researchers who analyze the outcome of gene editing in crops. For example, scientists at EAG Laboratories characterize genome editing in crop plants with a variety of techniques, including digestibility assays, ELISAs, mass spectrometry, real-time PCR (qPCR), and SDS-PAGE. As explained by LaHoma Easterwood, senior scientist and group leader at EAG Laboratories, “The techniques used help identify whether the crop plant is expressing the protein of interest, where the protein is being expressed and how abundantly it is being expressed.” Helping customers analyze gene-edited crops puts EAG Laboratories in a key role. “The testing and analysis of genome-editing technologies used for crop plants is an extremely important step in helping our customers identify which genes of interest will be beneficial to pursue,” says Easterwood. “The analyses that are performed help our customers gain a better understanding of how the gene of interest may affect other biological events in the crop plants as well as how it may benefit the consumer.”
By applying these advanced gene-editing mechanisms to crop plants, scientists can make it easier and more efficient to feed the world—and do less damage to the environment in the process.
References
[1] Boch, J, et al., “Breaking the code of DNA binding specificity of TAL-type III effectors,” Science, 326:1509-12, 2009. [PMID: 19933107]
[2] Moscou, MJ, Bogdanove, AJ, “A simple cipher governs DNA recognition by TAL effectors,” Science, 326:1501, 2009. [PMID: 19933106]
[3] Hendel, A, et al., “Quantifying genome-editing outcomes at endogenous loci with SMRT sequencing,” Cell Reports, 7:293-305, 2014. [PMID: 24685129]
[4] Li, T, et al., “TALEN utilization in rice genome modifications,” Methods, 69:9-16, 2014.
[PMID: 24680698]
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