When chromosomes break, the ends can join together in a number of ways, some of which can cause trouble. A new QA method could help researchers avoid making problematic breaks when using gene editing technologies like CRISPR.
Labs the world over are jumping onto the gene editing bandwagon (and into the inevitable patent arguments). And it’s hard to blame them. As these technologies have evolved over the last two decades starting with the zinc finger nucleases (ZFNs), followed by transcription activator-like effector nucleases (TALENs) and CRISPR—they’ve become ever more powerful and easier to use.
But one question keeps coming up: How precise are these systems? After all, a method that selectively mutates, deletes or swaps specific gene sequences (and now can even turn genes on) is only as good as its accuracy.
Algorithms can predict the likely “off-target” edits based on the target’s DNA sequence, but they’re based on limited data. “The algorithms are getting better,” says Richard Frock, PhD, a fellow in the laboratory of Frederick Alt, PhD, at Boston Children’s Hospital. “But you still worry about the one rare off-target effect that’s not predicted but falls in a coding region and totally debilitates a gene.”
Frock, Alt (who leads Boston Children’s Program in Cellular and Molecular Medicine, or PCMM), fellow Jiazhi Hu, PhD, and their collaborators recently turned a method first developed in Alt’s lab for studying broken chromosomes into a quality assurance tool for genome editing. As a bonus, the method—called high-throughput genome translocation sequencing (HTGTS)—also reveals the “collateral damage” gene editing methods might create in a cell’s genome, information that could help researchers make better choices when designing gene editing experiments. Full story »
Researchers discovered many small nuances in pluripotency states of stem cells by subjecting the cells to various perturbations and then analyzing each individual cell to observe all the different reactions to developmental cues within a stem cell colony. (Credit: Wyss Institute at Harvard University)
Stem cells offer great potential in biomedical engineering because they’re pluripotent—meaning they can multiply indefinitely and develop into any of the hundreds of different kinds of cells and tissues in the body. But in trying to tap these cells’ creative potential, it has so far been hard to pinpoint the precise biological mechanisms and genetic makeups that dictate how stem cells diverge on the path to development.
Part of the challenge, according to James Collins, PhD, a core faculty member at the Wyss Institute for Biologically Inspired Engineering, is that not all stem cells are created the same. “Stem cell colonies contain much variability between individual cells. This has been considered somewhat problematic for developing predictive approaches in stem cell engineering,” he says.
While at first glimmer, it could appear this would make predictive stem cell engineering more difficult, it might actually present an opportunity to exert even more programmable control over stem cell differentiation and development than was originally envisioned. “What was previously considered problematic variability could actually be beneficial to our ability to precisely control stem cells,” says Collins. Full story »
Neurons from patients could lead researchers to better drugs for chronic pain.
Chronic pain, affecting tens of millions of Americans alone, is debilitating and demoralizing. It has many causes, and in the worst cases, people become “hypersensitized”—their nervous systems fire off pain signals in response to very minor triggers.
CRISPR—a gene editing technology that lets researchers make precise mutations, deletions and even replacements in genomic DNA—is all the rage among genomic researchers right now. First discovered as a kind of genomic immune memory in bacteria, labs around the world are trying to leverage the technology for diseases ranging from malaria to sickle cell disease to Duchenne muscular dystrophy.
In a paper published yesterday in Cell Stem Cell, a team led by Derrick Rossi, PhD, of Boston Children’s Hospital, and Chad Cowan, PhD, of Massachusetts General Hospital, report a first for CRISPR: efficiently and precisely editing clinically relevant genes out of cells collected directly from people. Specifically, they applied CRISPR to human hematopoietic stem and progenitor cells (HSPCs) and T-cells.
“CRISPR has been used a lot for almost two years, and report after report note high efficacy in various cell lines. Nobody had yet reported on the efficacy or utility of CRISPR in primary blood stem cells,” says Rossi, whose lab is in the hospital’s Program in Cellular and Molecular Medicine. “But most researchers would agree that blood will be the first tissue targeted for gene editing-based therapies. You can take blood or stem cells out of a patient, edit them and transplant them back.”
The study also gave the team an opportunity to see just how accurate CRISPR’s cuts are. Their conclusion: It may be closer to being clinic-ready than we thought. Full story »
Dolly the sheep, the first mammalian example of successful somatic cell nuclear transfer. (Toni Barros/Wikimedia Commons)
We all remember Dolly the sheep, the first mammal to be born through a cloning technique called somatic cell nuclear transfer (SCNT). As with the thousands of other SCNT-cloned animals ranging from mice to mules, researchers created Dolly by using the nucleus from a grown animal’s cell to replace the nucleus of an egg cell from the same species.
The idea behind SCNT is that the egg’s cellular environment kicks the transferred nucleus’s genome into an embryonic state, giving rise to an animal genetically identical to the nucleus donor. SCNT is also a technique for generating embryonic stem cells for research purposes.
While researchers have accomplished SCNT in many animal species, it could work better than it does now. It took the scientists who cloned Dolly 277 tries before they got it right. To this day, SCNT efficiency—that is, the percent of nuclear transfers it takes generate a living animal—still hovers around 1 to 2 percent for mice, 5 to 20 percent in cows and 1 to 5 percent in other species. By comparison, the success rate in mice of in vitro fertilization (IVF) is around 50 percent.
Blood-forming hematopoietic stem cells (top) give rise to all blood and immune cell types. In children with SCID, the steps leading to immune cells are broken.
In the world of fatal congenital immunodeficiency diseases, good news is always welcome, because most patients die before their first birthday if not treated. Babies with severe combined immunodeficiency disease, aka SCID or the “bubble boy disease,” now have more hope for survival thanks to two pieces of good news.
Credit: Samantha Morris, PhD, Boston Children's Hospital
If you’ve lost your way on the Boston subway, you need only consult a map to find the best route to your destination. Now stem cell engineers have a similar map to guide the making of cells and tissues for disease modeling, drug testing and regenerative medicine. It’s a computer algorithm known as CellNet.
As in this map on the cover of Cell, a cell has many possible destinations or “fates,” and can arrive at them through three main stem cell engineering methods:
• reprogramming (dialing a specialized cell, such as a skin cell, back to a stem-like state with full tissue-making potential)
• differentiation (pushing a stem cell to become a particular cell type, such as a blood cell)
• direct conversion (changing one kind of specialized cell to another kind)
At TEDx Longwood this spring, Leonard Zon, MD, founder and director of the Stem Cell Program at Boston Children’s Hospital, took the stage. In his enthusiastic yet humble style, he took the audience on a journey that included time-lapse video of zebrafish embryos developing, a riff by Jay Leno and a comparison of stem cell “engraftment” to a college kid coming home after finals: “You sleep for three days, and on day 4, you wake up and you’re in your own bed.” Three takeaways:
1) Stem cells made from our own skin cells can help find new therapeutics. With the right handling, they themselves can be therapeutics, producing healthy muscle, insulin-secreting cells, pretty much anything we need. (So far, this has just been done in mice.)
2) Zebrafish, especially when they’re see-through, can teach us how stem cells work and can be used for mass screening of potential drugs. The Zon Lab boasts 300,000 of these aquarium fish, and can mount robust “clinical trials” with 100 fish per group.
A restored, clear cornea grown from ABCB5-positive limbal stem cells. (Image courtesy of the researchers)
Severe burns, chemical injury and certain diseases can cause blindness by clouding the eyes’ corneas and killing off a precious population of stem cells that help maintain them. In the past, doctors have tried to regrow corneal tissue by transplanting cells from limbal tissue—found at the border between the cornea and the white of the eye. But they didn’t know whether the tissue contained enough of the active ingredient: limbal stem cells.
How cancer research led to a regenerative treatment for blindness.
Results have therefore been mixed. “Limbal stem cells are very rare, and successful transplants are dependent on these rare cells,” says Bruce Ksander, PhD, of the Massachusetts Eye and Ear/Schepens Eye Research Institute. “If you have a limbal stem cell deficiency and receive a transplant that does not contain stem cells, the cornea will become opaque again.”
Limbal stem cells have been sought for over a decade. That’s where a “tracer” molecule called ABCB5—first studied in the context of cancer—comes in. Full story »
The fat cells shown in yellow are descended from transplanted human mesenchymal stem cells (green) inside of a mouse after co-transplantation. The red stain shows native mouse fat cells.(Courtesy Juan Melero-Martin)
Stem cell scientists had what first appeared to be an easy win for regenerative medicine when they discovered mesenchymal stem cells several decades ago. These cells, found in the bone marrow, can give rise to bone, fat and muscle tissue, and have been used in hundreds of clinical trials for tissue repair.
Uses range from tissue protection in heart attack and stroke to immune modification in multiple sclerosis and diabetes. Unfortunately, the results of these trials have been underwhelming. One challenge is that these stem cells don’t stick around in the body long enough to benefit the patient. Full story »