Focus on: Genome editing
NON-BROWNING MUSHROOMS
What is an "edited" mushroom?
Introduction
There's a new type of mushroom available. It doesn't look different than normal mushrooms, but it stays fresh and white much longer than other mushrooms. What's different about this mushroom? It's the product of a new, very precise process called genome editing. Scientists used genetic knowledge to change its genes and stop it from turning brown. How? Look deeper to explore…
What is an "edited" mushroom?
About the organism
The white button mushroom, Agaricus bisporus, is a type of fungus. The part we eat is the reproductive structure, you can think of it as a tiny"fruit", that grows out of the mycelium, the main body of the fungus. The button mushroom's mycelium is a tangled web of cells under the ground.

Fungi don't have seeds. Instead, they have spores, which are found on the gills under the button mushroom's cap. A young mushroom is tightly curved, with the spores nearly enclosed. As the mushroom ages, the cap flattens, exposing the gills and the spores darken.


The mushrooms we eat are the fruiting bodies of a giant underground mass called the mycelium.
A mushroom is a fungus, not a plant; it can't photosynthesize. So how does it get the energy to grow? The cells of the mushroom's mycelium secrete enzymes that break down the food sources surrounding it. Then the simple digested molecules are absorbed back into the mycelium to fuel its growth and reproduction. In an ecosystem, the button mushroom is a decomposer. It grows by utilizing the nutrients in organic matter like compost, cocoa hulls or even manure.
Button mushrooms require very different farming conditions than plants. Rather than regular soil, mushrooms need specially prepared compost in a dark, moist environment. Moisture is critical because mushrooms lack many of the water-conservation features that plants have—like seed coats and waxy surfaces. In the compost, the mushroom's spores can develop into mycelium, from which button mushrooms can grow and develop. The process, from preparing the compost, through growth, harvesting and shipment, takes farmers about four months. A productive mycelium can produce button mushrooms to harvest every few days.
Mushrooms bring many of the health benefits of vegetables—a food group they're commonly assigned to. Of course a mushroom is a fungus, and not a vegetable at all. But mushrooms are a good source of many vitamins and minerals—particularly B vitamins. They are a low-calorie, filling food with many health benefits.
Think and Apply
What does a mushroom farm look like?
Answer:
You won't see fields of mushrooms on a mushroom farm. Instead, mushrooms grow on special compost in damp, dark environments. There, their spores can develop into mycelium, from which the mushroom fruiting bodies grow.
More than 60% of the mushrooms in the United States grow on mushroom farms in southeastern Pennsylvania, where they are $550 million dollar industry. In the United States, California and Washington states also have substantial amounts of mushroom production. Mushrooms are also grown in 70 countries around the world, with more than 70% produced in China. Look closer to see what a mushroom farm looks like and how the mushrooms grow.
A mushroom farm looks different than a vegetable farm.
How Nutritious?
Button mushrooms are a low calorie, low sodium, fat-free food. Mushrooms are a good source of many different vitamins and minerals including phosphorous, the B vitamins, selenium, and copper.

Button mushrooms can be eaten raw or cooked. They have a mild flavor, that intensifies when the mushroom is cooked.

Think and Apply
There are many different B vitamins, each of which is an important metabolic cofactor or a precursor to a cofactor. Read the nutrition label and indicate whether mushrooms contain each of the following B vitamins: Thiamin (B1), Riboflavin (B2), Niacin (B3), Pantothenic acid (B5), Pyridoxine (B6), Biotin (B7), Folate (B9), Vitamin (B12)
Answer:
Mushrooms are a great source of riboflavin (20% of a recommended daily amount), and a good source of niacin and pantothenic acid (15% each). They contain thiamin and folate (4% each). Though pyridoxine and vitamin B12 don't show up on the label, mushrooms contain small amounts of both.
The Challenge
Reduce mushroom browning
Mushrooms can turn brown during storage. Why? Enzymes cause chemical reactions that change the color of the mushroom. The enzymes are controlled by the mushroom's genes. Could a small change to the mushroom's genes result in a big change for freshness and storage?
Can a small change to an important enzyme reduce waste of a perishable food?
The Solution
Modify the mushroom genome using CRISPR-Cas9


How could some simple changes to the DNA of the perishable button mushroom extend its shelf life? The solution uses a new technique called CRISPR, or more accurately CRISPR-CAS9, to make targeted changes to a gene involved in the chemical reactions that turn mushrooms brown.
Think and Apply
DNA figure for think and apply box—DD is creating
Use what you know of DNA, genes and proteins to explain how DNA changes can alter a trait like browning?
Answer:
DNA, or deoxyribonucleic acid, is found in every cell of every organism, be it an animal, plant, fungus, bacteria or archaebacteria. DNA is made of units called nucleotides, which each have a sugar, a phosphate group and a nitrogenous base (adenine, thymine, guanine, and cytosine). The DNA molecules are very long, but within them, certain smaller sections of nucleotides carry the instructions to make proteins; these sections of DNA are called genes.

Through the processes of transcription (to make RNA) and translation, proteins are made from the DNA code in a gene. Proteins are involved in the expression of traits.

Therefore, a change to the mushroom's DNA may change the protein or enzyme it encodes. A change to the right enzyme could affect a process like browning.
How can CRISPR-CAS9 change mushroom browning? The solution occurs in two parts. First we explore the CRISPR-CAS9 system to understand how it works and where it comes from. Then we see how Dr. Yang, of Penn State University, applied this new technology to edit the genes of the button mushroom to reduce browning. Editing a gene is tricky, but this new technique, based on a very old process, enables scientists to make very precise edits to targeted genes.
>Part 1: Where does CRISPR-Cas9 come from?

Bacteria can get viral infections just like we do. Not surprisingly, bacteria have evolved a way to protect themselves against viruses. The foundation of the "new" CIRSPR-CAS9 gene editing system is an ancient bacterial response to virus infection. Working much like parts of our immune system, the bacterium recognizes the viral DNA and makes a defense compound to destroy it.


Here is how it works: First, the virus lands on bacterial cell and injects its DNA into the cell (Step 1). Viruses don't have the cellular machinery to make copies of their own DNA. Instead, their DNA integrates into a host cell and uses the host to make copies of the virus's components. Next, the bacterium detects the viral DNA and actually incorporates it into a special section of its own bacterial DNA called CRISPR DNA, short for Clustered Regularly Interspaced Short Palindromic Repeats (Step 2).

A bacterial immune response offers a means to edit genes
The bacterium then transcribes the CRISPR DNA and produces two specialized types of RNA: CRISPR RNA (crRNA), which matches the viral DNA, and tracer RNA (Step 3). Both of those RNA pieces then form a complex with CAS9, an endonuclease, or an enzyme that can cut DNA (Step 4). The guide crRNA, now part of the CAS complex, finds the matching sequence on the viral DNA (Step 5). The CAS protein-RNA complex unwinds and cuts the viral DNA (Step 6). Because the viral DNA is cut, it can no longer infect the bacterial cell.
Think and Apply
Now that you understand the bacterial system, imagine how the CAS endonuclease paired with some different RNA could be used to edit genes of other organisms.
Answer:
The key elements of this system can work in any living cell not just bacteria. Therefore, scientists can use this system to edit cells of any organism.
When DNA is damaged, a cell can naturally repair the break. The genome editing process takes advantage of natural repair mechanisms. By introducing breakage into a formerly functional gene, CRISPR-Cas9 can disable genes. With a few small tweaks, the same system can be used to introduce functional copies of broken genes.
MIT Gene Editing Video
Genome editing takes advantage of natural processes to disable or change genes in living cells.

Part 2: How was CRISPR-Cas9 used in mushrooms?

Browning in a white button mushroom is caused by chemical reactions of the polyphenol oxidase enzyme. Plant pathologist Yinong Yang at Penn State University recognized that the CRISPR-CAS9 system could potentially be used to disable a gene that produces polyphenol oxidase. As a result, less PPO would be produced by the mushroom, which means the mushroom doesn't turn brown as quickly. This modified mushroom contains no foreign DNA in its genome; it simply has a gene disabled.

Watch the video below to learn more about what Dr. Yang did.

Meet Dr. Yang
Dr. Yinong Yang at Penn State decided to use CRISPR-Cas9 to knock out the PPO gene in button mushrooms.
Conclusion
As you have seen, CRISPR-CAS9 is a powerful gene-editing tool. The combination of guide RNA and an endonuclease can be used to either disable a gene by cutting target DNA or to insert new DNA into a sequence after it is cut. CRISPR-CAS9 is speeding up gene editing and opening up new possibilities for tailoring plant traits.

To reduce browning in button mushrooms, Dr. Yang used the CRISPR-CAS9 system to disable one of the polyphenol oxidase genes. By reducing the amount of the PPO enzyme produced, he created a mushroom much less susceptible to browning. Less browning on a mushroom means it can be mechanically harvested without damage and gives it a longer shelf-life, which saves money and reduces waste.

The mushroom is not yet on store shelves, but Dr. Yang submitted the mushroom to the USDA (UnitedStates Department of Agriculture) for review in Spring 2016 and was given the thumbs up. Because Dr. Yang's use of the CRISPR-CAS9 technology simply removed a gene and the resulting mushrooms do not contain any new, introduced genetic material, the USDA decided the agency did not have to regulate this PPO-reduced mushroom. Other agencies like the FDA (Foodand Drug Administration) will evaluate its safety.
In April 2016, the USDA commented that since the CRISPR-modified mushroom did not contain any new, introduced genetic material the USDA did not need to regulate it. Other agencies like the Food and Drug Administration will still evaluate its safety.
Left
Right
This new technique leaves us with many new questions. What other foods will scientists modify using the CRIPSR-CAS9 gene editing system? What genes will they disable? Will scientists use CRISPR-CAS9 to insert or otherwise modify genes? And what will that mean for regulation? Only the future will tell. Follow the news for food stories and see what the future of food holds!
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