Posted on by Dr. Francis Collins
If used to make non-heritable genetic changes, CRISPR gene-editing technology holds tremendous promise for treating or curing a wide range of devastating disorders, including sickle cell disease, vision loss, and muscular dystrophy. Early efforts to deliver CRISPR-based therapies to affected tissues in a patient’s body typically have involved packing the gene-editing tools into viral vectors, which may cause unwanted immune reactions and other adverse effects.
Now, NIH-supported researchers have developed an alternative CRISPR delivery system: nanocapsules. Not only do these tiny, synthetic capsules appear to pose a lower risk of side effects, they can be precisely customized to deliver their gene-editing payloads to many different types of cells or tissues in the body, which can be extremely tough to do with a virus. Another advantage of these gene-editing nanocapsules is that they can be freeze-dried into a powder that’s easier than viral systems to transport, store, and administer at different doses.
In findings published in Nature Nanotechnology , researchers, led by Shaoqin Gong and Krishanu Saha, University of Wisconsin-Madison, developed the nanocapsules with specific design criteria in mind. They would need to be extremely small, about the size of a small virus, for easy entry into cells. Their surface would need to be adaptable for targeting different cell types. They also had to be highly stable in the bloodstream and yet easily degraded to release their contents once inside a cell.
After much hard work in the lab, they created their prototype. It features a thin polymer shell that’s easily decorated with peptides or other ingredients to target the nanocapsule to a predetermined cell type.
At just 25 nanometers in diameter, each nanocapsule still has room to carry cargo. That cargo includes a single CRISPR/Cas9 scissor-like enzyme for snipping DNA and a guide RNA that directs it to the right spot in the genome for editing.
In the bloodstream, the nanocapsules remain fully intact. But, once inside a cell, their polymer shells quickly disintegrate and release the gene-editing payload. How is this possible? The crosslinking molecules that hold the polymer together immediately degrade in the presence of another molecule, called glutathione, which is found at high levels inside cells.
The studies showed that human cells grown in the lab readily engulf and take the gene-editing nanocapsules into bubble-like endosomes. Their gene-editing contents are then released into the cytoplasm where they can begin making their way to a cell’s nucleus within a few hours.
Further study in lab dishes showed that nanocapsule delivery of CRISPR led to precise gene editing of up to about 80 percent of human cells with little sign of toxicity. The gene-editing nanocapsules also retained their potency even after they were freeze-dried and reconstituted.
But would the nanocapsules work in a living system? To find out, the researchers turned to mice, targeting their nanocapsules to skeletal muscle and tissue in the retina at the back of eye. Their studies showed that nanocapsules injected into muscle or the tight subretinal space led to efficient gene editing. In the eye, the nanocapsules worked especially well in editing retinal cells when they were decorated with a chemical ingredient known to bind an important retinal protein.
Based on their initial results, the researchers anticipate that their delivery system could reach most cells and tissues for virtually any gene-editing application. In fact, they are now exploring the potential of their nanocapsules for editing genes within brain tissue.
I’m also pleased to note that Gong and Saha’s team is part of a nationwide consortium on genome editing supported by NIH’s recently launched Somatic Cell Genome Editing program. This program is dedicated to translating breakthroughs in gene editing into treatments for as many genetic diseases as possible. So, we can all look forward to many more advances like this one.
 A biodegradable nanocapsule delivers a Cas9 ribonucleoprotein complex for in vivo genome editing. Chen G, Abdeen AA, Wang Y, Shahi PK, Robertson S, Xie R, Suzuki M, Pattnaik BR, Saha K, Gong S. Nat Nanotechnol. 2019 Sep 9.
Saha Lab (University of Wisconsin-Madison)
Shaoqin (Sarah) Gong (University of Wisconsin-Madison)
NIH Support: National Eye Institute; National Institute of General Medical Sciences; National Institute of Neurological Disorders and Stroke; National Heart, Lung, and Blood Institute; Common Fund
Posted on by Dr. Francis Collins
Nanoparticles hold great promise for delivering next-generation therapeutics, including those based on CRISPR gene editing tools. The challenge is how to guide these tiny particles through the bloodstream and into the right target tissues. Now, scientists are enlisting some surprising partners in this quest: magnetic bacteria!
First a bit of background. Discovered in the 1960s during studies of bog sediments, “magnetotactic” bacteria contain magnetic, iron-rich particles that enable them to orient themselves to the Earth’s magnetic fields. To explore the potential of these microbes for targeted delivery of nanoparticles, the NIH-funded researchers devised the ingenious system you see in this fluorescence microscopy video. This system features a model blood vessel filled with a liquid that contains both fluorescently-tagged nanoparticles (red) and large swarms of a type of magnetic bacteria called Magnetospirillum magneticum (not visible).
At the touch of a button that rotates external magnetic fields, researchers can wirelessly control the direction in which the bacteria move through the liquid—up, down, left, right, and even “freestyle.” And—get this—the flow created by the synchronized swimming of all these bacteria pushes along any nearby nanoparticles in the same direction, even without any physical contact between the two. In fact, the researchers have found that this bacteria-guided system delivers nanoparticles into target model tissues three times faster than a similar system lacking such bacteria.
How did anyone ever dream this up? Most previous attempts to get nanoparticle-based therapies into diseased tissues have relied on simple diffusion or molecular targeting methods. Because those approaches are not always ideal, NIH-funded researchers Sangeeta Bhatia, Massachusetts Institute of Technology, Cambridge, MA, and Simone Schürle, formerly of MIT and now ETH Zurich, asked themselves: Could magnetic forces be used to propel nanoparticles through the bloodstream?
As a graduate student at ETH Zurich, Schürle had worked to develop and study tiny magnetic robots, each about the size of a cell. Those microbots, called artificial bacterial flagella (ABF), were designed to replicate the movements of bacteria, relying on miniature flagellum-like propellers to move them along in corkscrew-like fashion.
In a study published recently in Science Advances, the researchers found that the miniature robots worked as hoped in tests within a model blood vessel . Using magnets to propel a single microbot, the researchers found that 200-nanometer-sized polystyrene balls penetrated twice as far into a model tissue as they did without the aid of the magnet-driven forces.
At the same time, others in the Bhatia lab were developing bacteria that could be used to deliver cancer-fighting drugs. Schürle and Bhatia wished they could direct those microbial swarms using magnets as they could with the microbots. That’s when they learned about the potential of M. magneticum and developed the experimental system demonstrated in the video above.
The researchers’ next step will be to test their magnetic approach to drug delivery in a mouse model. Ultimately, they think their innovative strategy holds promise for delivering nanoparticles carrying a wide range of therapeutic payloads right to a tumor, infection, or other diseased tissue. It’s yet another example of how basic research combined with outside-the-box thinking can lead to surprisingly creative solutions with real potential to improve human health.
 Synthetic and living micropropellers for convection-enhanced nanoparticle transport. Schürle S, Soleimany AP, Yeh T, Anand GM, Häberli M, Fleming HE, Mirkhani N, Qiu F, Hauert S, Wang X, Nelson BJ, Bhatia SN. Sci Adv. 2019 Apr 26;5(4):eaav4803.
What are genome editing and CRISPR-Cas9? (National Library of Medicine/NIH)
Sangeeta Bhatia (Massachusetts Institute of Technology, Cambridge, MA)
Simone Schürle-Finke (ETH Zurich, Switzerland)
NIH Support: National Cancer Institute; National Institute of General Medical Sciences
Posted on by Dr. Francis Collins
Technological advances with potential for improving human health sometimes come from the most unexpected places. An intriguing example is an electricity-conducting biological nanowire that holds promise for powering miniaturized pacemakers and other implantable electronic devices.
The nanowires come from a bacterium called Geobacter sulfurreducens, shown in the electron micrograph above. This rod-shaped microbe (white) was discovered two decades ago in soil collected from an unlikely place: a ditch outside of Norman, Oklahoma. The bug can conduct electricity along its arm-like appendages, and, in the hydrocarbon-contaminated, oxygen-depleted soil in which it lives, such electrical inputs and outputs are essentially the equivalent of breathing.
Scientists fascinated with G. sulfurreducens thought that its electricity had to be flowing through well-studied microbial appendages called pili. But, as the atomic structure of these nanowires (multi-colors, foreground) now reveals, these nanowires aren’t pili at all! Instead, the bacteria have manufactured unique submicroscopic arm-like structures. These arms consist of long, repetitive chains of a unique protein, each surrounding a core of iron-containing molecules.
The surprising discovery, published in the journal Cell, was made by an NIH-funded team involving Edward Egelman, University of Virginia Health System, Charlottesville. Egelman’s lab has had a long interest in what’s called a type 4 pili. These strong, adhering appendages help certain infectious bacteria enter tissues and make people sick. In fact, they enable bugs like Neisseria meningitidis to cross the blood-brain barrier and cause potentially deadly bacterial meningitis. While other researchers had proposed that those same type 4 pili allowed G. sulfurreducens to conduct electricity, Egelman wasn’t so sure.
So, he took advantage of recent advances in cryo-electron microscopy, which involves flash-freezing molecules at extremely low temperatures before bombarding them with electrons to capture their images with a special camera. The cryo-EM images allowed his team to nail down the atomic structure of the nanowires, now called OmcS filaments.
Using those images and sophisticated bioinformatics, Egelman and team determined that OmcS proteins uniquely fit into the nanowires’ long repetitive chains, spacing their iron-bearing cores at regular intervals to transfer electrons and convey electricity. In fact, bacteria unable to produce OmcS proteins make filaments that conduct electricity 100 times less efficiently.
With these cryo-EM structures in hand, Egelman says his team will continue to explore their conductive properties. Such knowledge might someday be used to build biologically-inspired nanowires, measuring 1/100,000th the width of a human hair, to connect miniature electronic devices directly to living tissues. This is one more example of how nature’s ability to invent is pretty breathtaking—surely one wouldn’t have predicted the discovery of nanowires in a bacterium that lives in contaminated ditches.
 Structure of Microbial Nanowires Reveals Stacked Hemes that Transport Electrons over Micrometers. Wang F, Gu Y, O’Brien JP, Yi SM, Yalcin SE, Srikanth V, Shen C, Vu D, Ing NL, Hochbaum AI, Egelman EH, Malvankar NS. Cell. 2019 Apr 4;177(2):361-369.
Electroactive microorganisms in bioelectrochemical systems. Logan BE, Rossi R, Ragab A, Saikaly PE. Nat Rev Microbiol. 2019 May;17(5):307-319.
High Resolution Electron Microscopy (National Cancer Institute/NIH)
Egelman Lab (University of Virginia, Charlottesville)
NIH Support: National Institute of General Medical Sciences; National Institute of Allergy and Infectious Diseases; Common Fund
Posted on by Dr. Francis Collins
Using a screwdriver on the tiny microcircuits arrayed inside a computer hard drive can be a real eye strain. Even more challenging is building the microcircuits or other electronic components at the nanoscale, one-billionth of a meter or less.
That’s why researchers are always on the lookout for new tools to help them work on such a minute scale. But some of these incredibly tiny tools and scaffolds can derive from very unexpected sources.
As published in the journal Science, an NIH-funded team has developed a technique called implosion fabrication to build impressively small and intricate components on the nanoscale . Its secret ingredient: water-swollen gels that you’d find in a baby’s disposable diaper.
A baby’s disposable diaper? If that sounds familiar, my blog highlighted a related technique called expansion microscopy a few years ago that uses water-swollen gels that are generated from a compound used in diapers called sodium polyacrylate.
The previously-reported microscopy technique, from the lab of Edward Boyden, Massachusetts Institute of Technology, Cambridge, embeds biological samples in a fine web of sodium polyacrylate. When water is added, the gel expands, blowing up the specimen to 100 times its original size. This groundbreaking technique, called expansion microscopy, has enabled labs around the world to use conventional microscopes for high-resolution, nanoscale imaging.
In the latest work, Boyden’s team, including co-first authors Daniel Oran and Samuel Rodriques, asked a simple question: What would happen if they applied the sample preparation technique used for expansion microscopy—only in reverse?
To find out, Boyden’s team created millimeter-sized blocks of the super-absorbent sodium polyacrylate diaper compound. After using a nifty trick for attaching molecular anchors in a 3D pattern, they dehydrated the gel and voila! The structures imploded and shrank down to one-thousandth their original size, while holding their 3D shape.
During the process, they can add to the anchors a range of functional molecules or elements. These include DNA, nanoparticles, semiconductors, or almost anything that’s needed.
While more work is needed to perfect the new technique, the researchers have already shown it can create objects one cubic millimeter in size, engineered to include intricate details down to about 50 nanometers. For comparison, a virus is about 30 to 50 nanometers.
These latest findings come as a reminder that advances in biomedicine often lead in wonderful and unexpected new directions. Out of the NIH-funded efforts related to The Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative, members of the Boyden Lab wanted to see the brain better using basic microscopes. Now, we have a widely-applicable promising new approach to nanofabrication.
 3D nanofabrication by volumetric deposition and controlled shrinkage of patterned scaffolds. Oran D, Rodriques SG, Gao R, Asano S, Skylar-Scott MA, Chen F, Tillberg PW, Marblestone AH, Boyden ES. Science. 2018 Dec 14;362(6420):1281-1285.
Size of the Nanoscale (Nano.gov)
Synthetic Neurobiology Group, Ed Boyden (MIT, Cambridge, MA)
NIH Support: Common Fund; National Institute of Mental Health; National Institute of Biomedical Imaging and Bioengineering; National Human Genome Research Institute; National Institute on Drug Abuse; National Institute of Neurological Disorders and Stroke
Posted on by Dr. Francis Collins
Check out the world’s smallest board game, a nanoscale match of tic-tac-toe being played out in a test tube with X’s and O’s made of DNA. But the innovative approach you see demonstrated in this video is much more than fun and games. Ultimately, researchers hope to use this technology to build tiny DNA machines for a wide variety of biomedical applications.
Here’s how it works. By combining two relatively recent technologies, an NIH-funded team led by Lulu Qian, California Institute of Technology, Pasadena, CA, created a “swapping mechanism” that programs dynamic interactions between complex DNA nanostructures . The approach takes advantage of DNA’s modular structure, along with its tendency to self-assemble, based on the ability of the four letters of DNA’s chemical alphabet to pair up in an orderly fashion, A to T and C to G.
To make each of the X or O tiles in this game (displayed here in an animated cartoon version), researchers started with a single, long strand of DNA and many much shorter strands, called staples. When the sequence of DNA letters in each of those components is arranged just right, the longer strand will fold up into the desired 2D or 3D shape. This technique is called DNA origami because of its similarity to the ancient art of Japanese paper folding.
In the early days of DNA origami, researchers showed the technique could be used to produce miniature 2D images, such as a smiley face . Last year, the Caltech group got more sophisticated—using DNA origami to produce the world’s smallest reproduction of the Mona Lisa .
In the latest work, published in Nature Communications, Qian, Philip Petersen and Grigory Tikhomirov first mixed up a solution of nine blank DNA origami tiles in a test tube. Those DNA tiles assembled themselves into a tic-tac-toe grid. Next, two players took turns adding one of nine X or O DNA tiles into the solution. Each of the game pieces was programmed precisely to swap out only one of the tile positions on the original, blank grid, based on the DNA sequences positioned along its edges.
When the first match was over, player X had won! More importantly for future biomedical applications, the original, blank grid had been fully reconfigured into a new structure, built of all-new, DNA-constructed components. That achievement shows not only can researchers use DNA to build miniature objects, they can also use DNA to repair or reconfigure such objects.
Of course, the ultimate aim of this research isn’t to build games or reproduce famous works of art. Qian wants to see her DNA techniques used to produce tiny automated machines, capable of performing basic tasks on a molecular scale. In fact, her team already has used a similar approach to build nano-sized DNA robots, programmed to sort molecules in much the same way that a person might sort laundry . Such robots may prove useful in miniaturized approaches to drug discovery, development, manufacture, and/or delivery.
Another goal of the Caltech team is to demonstrate to the scientific community what’s possible with this leading-edge technology, in hopes that other researchers will pick up their innovative tools for their own applications. That would be a win-win for us all.
 Information-based autonomous reconfiguration in systems of DNA nanostructures. Petersen P, Tikhomirov G, Qian L. Nat Commun. 2018 Dec 18;9(1):5362
 Folding DNA to create nanoscale shapes and patterns. Rothemund PW. Nature. 2006 Mar 16;440(7082):297-302.
 Fractal assembly of micrometre-scale DNA origami arrays with arbitrary patterns. Tikhomirov G, Petersen P, Qian L. Nature. 2017 Dec 6;552(7683):67-71.
 A cargo-sorting DNA robot. Thubagere AJ, Li W, Johnson RF, Chen Z, Doroudi S, Lee YL, Izatt G, Wittman S, Srinivas N, Woods D, Winfree E, Qian L. Science. 2017 Sep 15;357(6356).
Paul Rothemund—DNA Origami: Folded DNA as a Building Material for Molecular Devices (Cal Tech, Pasadena)
The World’s Smallest Mona Lisa (Caltech)
Qian Lab (Caltech, Pasadena, CA)
NIH Support: National Institute of General Medical Sciences