CRISPR CRITTERS
New CRISPR-combo boosts genome editing power in plants
UMD researchers develop the latest advances to CRISPR with a method to edit and regulate multiple genes simultaneously.
Peer-Reviewed PublicationIMAGE: ON THE LEFT: POPLAR SPROUTS CONTAINING CRSPR-EDITED GENE. ON THE RIGHT, POPLAR SPROUTS WITH THE SAME CRSPR EDIT PLUS GENE ENHANCEMENTS TO IMPROVE GROWTH view more
CREDIT: UMD
Ten years ago, a new technology called CRISPR-CAS9, made it possible for scientists to change the genetic code of living organisms. As revolutionary as it was, the tool had its limitations. Like the first cell phones that could only perform one function, the original CRISPR method can perform one function: removing or replacing genes in a genetic sequence. Later iterations of CRISPR were developed for another function that allowed scientists to change gene expression by turning them on or off, without removing them from the genome. But each of these functions could only be performed independently in plants.
Now, scientists from the University of Maryland College of Agriculture and Natural Resources, have developed CRISPR-Combo, a method to edit multiple genes in plants while simultaneously changing the expression of other genes. This new tool will enable genetic engineering combinations that work together to boost functionality and improve breeding of new crops.
“The possibilities are really limitless in terms of the traits that can be combined,” said Yiping Qi, an associate professor in the Department of Plant Science and Landscape Architecture and co-author of the study. “But what is really exciting is that CRISPR-Combo introduces a level of sophistication to genetic engineering in plants that we haven’t had before.”
The new research appears in the May, 2022, issue of the journal Nature Plants.
The benefits of manipulating more than one gene at a time can far outweigh the benefits of any one manipulation on its own. For example, imagine a blight raging through wheat fields, threatening farmer livelihoods and food security. If scientists could remove a gene from the wheat that makes it susceptible to the blight and simultaneously turn on genes that shorten the plant’s life cycle and increase seed production, they could rapidly produce blight-resistant wheat before the disease had the chance to do too much damage.
That’s the type of engineering Qi and his team demonstrated in four different phases of experimentation.
Step One: proving the concept:
Qi and his team had previously developed new CRISPR methods to regulate gene expression in plants, and to edit multiple genes at the same time. But to develop CRISPR-Combo, they had to establish that they could perform both of those genetic engineering functions in parallel without negative consequences. In this new paper, they demonstrated that using tomato and rice cells,.
“As a proof of concept, we showed that we could knock out gene A and upregulate, or activate, gene B successfully, without accidentally crossing over and knocking out gene B or upregulating Gene A,” Qi said.
Then Qi and his colleagues tested CRISPR-Combo on a flowering plant called rockcress (ArabidopsisI), which is often used by researchers as a model for staple crops like corn and wheat. The researchers edited a gene that makes the plant more resistant to herbicides while activating a gene that causes early flowering, which produces seeds more quickly. The result was an herbicide-resistant rockcress plant that yielded eight generations in one year rather than the ordinary four.
More Efficient Engineering
For their third experiment, the team demonstrated how CRISPR-Combo could improve efficiency in plant breeding using tissue cultures from poplar trees. Breeding programs to develop new varieties of plants generally use tissue cultures rather than seeds—consider how a plant can regrow roots and leaves from a single stalk planted in the soil. Scientists genetically modify stem cells that have the ability to grow into full plants, and when those plants mature and produce seeds, the seeds will carry on the genetic modifications made to the stem cells.
Some plants are better at regenerating from tissue cultures than others, which makes this step the single largest bottleneck in genetic engineering of crops. For some plants the success rate is just 1%.
Qi and his team addressed the bottleneck by first editing a few traits in poplar cells, then activating three genes that promote plant tissue regeneration.
“We showed in poplars that our new method could offer a solution to the tissue regeneration bottleneck, dramatically increasing the efficiency of genetic engineering,” Qi said.
Hormone-Free Short Cut
Currently, growing genetically engineered plants from tissue cultures requires the addition of growth hormones, which activate growth promoting genes. The research team shortcut this process in rice by directly activating these genes with CRISPR-Combo. The result was gene-edited rice from tissue cultures that did not require hormone supplementation. Qi and his colleagues found that tissue cultures grown with their method expressed more of the edited gene than tissue grown using hormones.
“This method results in a highly efficient genome editing process,” Qi said.
Now that the team has demonstrated their CRISPR-Combo method works in a variety of plants for multiple purposes, they intend to conduct experiments in citrus, carrots and potatoes to test its viability in a fruit, vegetable and staple crop. They are also working to create an herbicide resistant golden rice with enhanced nutritional content and red rice with increased antioxidants.
Other co-authors on the research paper from UMD include Associate Professor Gary Coleman, post-doctoral associates Changtian Pan and Gen Li, Post-doctoral scholar Filiz Gurel, graduate students Yanhao Cheng, Aimee A. Malzahn and Simon Sretenovic, Laboratory trainee and high school student Benjamin Leyson.
This work was supported by NSF Plant Genome Research Program (award nos. IOS-1758745 and IOS-2029889), the USDA-NIFA (award nos. 2020-33522-32274 and 2019-67013-29197), the USDA-AFRI Agricultural Innovations Through Gene Editing Program (award no. 2021-67013-34554), Maryland Innovation Initiative Funding (award no. 1120-012_2), the USDA McIntire-Stennis project (award no. MD-PSLA-20006), NRT-INFEWS: UMD Global STEWARDS through the NSF National Research Traineeship Program (award no. 1828910) and the Foundation for Food and Agriculture Research. This story does not necessarily reflect the views of these organizations.
JOURNAL
Nature Plants
METHOD OF RESEARCH
Experimental study
SUBJECT OF RESEARCH
Not applicable
ARTICLE TITLE
Gene-edited tomatoes could be a new
source of vitamin D
IMAGE: DR JIE LI EXAMINES VITAMIN D ENRICHED TOMATOES view more
CREDIT: PHIL ROBINSON
Tomatoes gene-edited to produce vitamin D, the sunshine vitamin, could be a simple and sustainable innovation to address a global health problem.
Researchers used gene editing to turn off a specific molecule in the plant’s genome which increased provitamin D3 in both the fruit and leaves of tomato plants. It was then converted to vitamin D3 through exposure to UVB light.
Vitamin D is created in our bodies after skin’s exposure to UVB light, but the major source is food. This new biofortified crop could help millions of people with vitamin D insufficiency, a growing issue linked to higher risk of cancer, dementia, and many leading causes of mortality. Studies have also shown that vitamin D insufficiency is linked to increased severity of infection by Covid-19.
Tomatoes naturally contain one of the building blocks of vitamin D3, called provitamin D3 or 7-dehydrocholesterol (7-DHC), in their leaves at very low levels. Provitamin D3, does not normally accumulate in ripe tomato fruits.
Researchers in Professor Cathie Martin’s group at the John Innes Centre used CRISPR-Cas9 gene editing to make revisions to the genetic code of tomato plants so that provitamin D3 accumulates in the tomato fruit. The leaves of the edited plants contained up to 600 ug of provitamin D3 per gram of dry weight. The recommended daily intake of vitamin d is 10 ug for adults.
When growing tomatoes leaves are usually waste material, but those of the edited plants could be used for the manufacture of vegan-friendly vitamin D3 supplements, or for food fortification.
“We’ve shown that you can biofortify tomatoes with provitamin D3 using gene editing, which means tomatoes could be developed as a plant-based, sustainable source of vitamin D3,” said Professor Cathie Martin, corresponding author of the study which appears in Nature Plants.
“Forty percent of Europeans have vitamin D insufficiency and so do one billion people world-wide. We are not only addressing a huge health problem, but are helping producers, because tomato leaves which currently go to waste, could be used to make supplements from the gene-edited lines.”
Previous research has studied the biochemical pathway of how 7-DHC is used in the fruit to make molecules and found that a particular enzyme Sl7-DR2 is responsible for converting this into other molecules.
To take advantage of this the researchers used CRISPR-Cas 9 to switch off this Sl7-DR2 enzyme in tomato so that the 7DHC accumulates in the tomato fruit.
They measured how much 7-DHC there was in the leaves and fruits of these edited tomato plants and found that there was a substantial increase in levels of 7-DHC in both the leaves and fruit of the edited plants.
The 7-DHC accumulates in both the flesh and peel of the tomatoes.
The researchers then tested whether the 7-DHC in the edited plants could be converted to vitamin D3 by shining UVB light on leaves and sliced fruit for 1 hour. They found that it did and was highly effective.
After treatment with UVB light to turn the 7-DHC into Vitamin D3, one tomato contained the equivalent levels of vitamin D as two medium sized eggs or 28g tuna – which are both recommended dietary sources of vitamin D.
The study says that vitamin D in ripe fruit might be increased further by extended exposure to UVB, for example during sun-drying.
Blocking the enzyme in the tomato had no effect on growth, development or yield of the tomato plants. Other closely related plants such as aubergine, potato and pepper have the same biochemical pathway so the method could be applied across these vegetable crops.
Earlier this month the UK Government announced an official review to examine whether food and drink should be fortified with vitamin D to address health inequalities.
Most foods contain little vitamin D and plants are generally very poor sources. Vitamin D3 is the most bioavailable form of vitamin D and is produced in the body when the skin is exposed to sunlight. In winter and in higher latitudes people need to get vitamin D from their diet or supplements because the sun is not strong enough for the body to produce it naturally.
First author of the study Dr Jie Li said: “The Covid-19 pandemic has helped to highlight the issue of vitamin D insufficiency and its impact on our immune function and general health. The provitamin D enriched tomatoes we have produced offer a much-needed plant-based source of the sunshine vitamin. That is great news for people adopting a plant-rich, vegetarian or vegan diet, and for the growing number of people worldwide suffering from the problem of vitamin D insufficiency.”
‘Biofortified tomatoes provide a new route to vitamin D sufficiency’ appears in Nature Plants.
JOURNAL
Nature Plants
METHOD OF RESEARCH
Experimental study
SUBJECT OF RESEARCH
Not applicable
ARTICLE TITLE
Biofortified tomatoes provide a new route to vitamin D sufficiency
ARTICLE PUBLICATION DATE
23-May-2022
