Scientists Boost Gene-Editing Tools To New Heights In Human Stem Cells

February 3, 2020

During the past decade, the gene editing tool CRISPR has transformed biology and opened up hopeful avenues to correct deadly inherited diseases. Now, the first human clinical trials using CRISPR have begun in the hopes of curing diseases by taking damaged cells out of patient and repairing and replacing them.

But along with this promise of regenerative, personalized medicine, CRISPR also can have significant safety limitations. CRISPR may not edit in the right place (so-called off-target gene effects) or may not be terribly efficient (successful editing may only be achieved about 10% of the time for every available cell target).

These limitations have frustrated scientists such as Arizona State University’s David Brafman, a cell bioengineer. Brafman initially hopes to use gene editing to get at the heart of the causes of neurodegenerative diseases like Alzheimer’s.

“We study neurodegenerative diseases like Alzheimer’s and use stem cells to study specific mutations or risk factors associated with Alzheimer’s disease,” said Brafman, a biomedical engineering faculty member in ASU’s Ira A. Fulton Schools of Engineering. “We are not necessarily a gene-editing tool development lab, but we were running into difficulty generating stem cell lines by using a traditional CRISPR-based editing approach. For reasons that are still unknown, stem cells are really resistant to that sort of genetic modification.”

Green light means go

Now, Brafman, using a new update to the CRISPR base editing technology originally developed in the lab of David Liu at Harvard, has vastly outperformed previous efforts by making highly accurate, single-DNA base editing with an efficiency of up to 90% of human stem cells. The results were published in the journal Stem Cell Reports.

“Previously, with CRISPR, it’s just been a random guess,” said Brafman. “And so, if you are picking at random stem cells and the efficiency is low, you’ll likely get only 10% or 5% because you have no idea if the edits have been made — the cell isn’t telling you.”

David Brafman

Brafman’s lab has developed a new TREE (Transient Reporter for Editing Enrichment) method, which allows for bulk enrichment of DNA base-edited cell populations — and for the first time, high efficiency in human stem cell lines.

“Most of the studies are done in immortalized cell lines or cancer cell lines, which are relatively easy to edit,” Brafman said. “This is the first example of using base editors in pluripotent stem cells, which is a very valuable cell population to genetically modify. We envision this method will have important implications for the use of human stem cell lines in developmental biology, disease modeling, drug screening and tissue engineering applications.”

Last year, they had shown that their TREE approach can work in human cell lines, but wanted to further push the technology to find a way to rapidly and efficiently edit human stem cell lines.

Unlike CRISPR, which cuts across both DNA stands, TREE only makes a single strand nick in DNA. For example, when a single DNA base is successfully edited from a C to a T, a protein gives off a signal, turning from blue to green.

“Now, if a cell is telling you, ‘If I’m glowing green I have a 90% chance of being edited you are going to have better luck identifying edited populations,” Brafman said. “Then, you can exclude all of the cells that are not edited. We isolate single cells that are glowing green, then grow those up into clonal populations that you are able to expand indefinitely.”

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https://scienceblog.com/513871/scientists-boost-gene-editing-tools-to-new-heights-in-human-stem-cells/

Scientists from The University of Texas at Austin report in the journal Science that they have developed a new strategy to protect honey bees from a deadly trend known as colony collapse: genetically engineered strains of bacteria.

An increasing number of honey bee colonies in the U.S. have seen the dwindling of their adult bees. According to a national survey, beekeepers lost nearly 40% of their honey bee colonies last winter, the highest rate reported since the survey began 13 years ago.

The engineered bacteria live in the guts of honey bees and act as biological factories, pumping out medicines protecting the bees against two major causes of colony collapse: Varroa mites and deformed wing virus. The researchers believe their method could one day scale up for agricultural use because the engineered bacteria are easy to grow, inoculating the bees is straightforward and the engineered bacteria are unlikely to spread beyond bees.

"It has direct implications for bee health," said Nancy Moran, a professor of integrative biology and the primary investigator on the study.

"This is the first time anyone has improved the health of bees by genetically engineering their microbiome," added Sean Leonard, a graduate student and first author of the study.

Varroa mites and deformed wing virus often come together; as the mites feed on bees, they can spread the virus, while also weakening the bees and making them more vulnerable to pathogens in the environment.

To address each problem, the team engineered one strain of bacteria to target the virus and another for the mites. Compared with control bees, the bees treated with the strain of bacteria targeting the virus were 36.5% more likely to survive to day 10. Meanwhile, Varroa mites feeding on another set of bees treated with the mite-targeting strain of bacteria were about 70% more likely to die by day 10 than mites feeding on control bees.

According to the American Beekeeping Federation, honey bees contribute nearly $20 billion each year to the value of U.S. crop production, and they play an enormous role in global food production. Without honey bees, dozens of crops, from almonds to berries to broccoli, would either vanish or produce significantly less food.

Like humans, honey bees have an ecosystem of bacteria in their guts called a microbiome and also an antiviral defense mechanism called RNA interference (RNAi) that helps the body fight off certain viruses, called RNA viruses. When an RNA virus is introduced, it produces molecules called double-stranded RNAs that a healthy cell detects, triggering an RNAi immune response.

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https://www.sciencedaily.com/releases/2020/01/200130144335.htm