Tag Archives: cells

The CRISPR ((clustered regularly interspaced short palindromic repeats)-CAS9 gene-editing technique may cause new genetic damage kerfuffle

Setting the stage

Not unexpectedly, CRISPR-Cas9  or clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 can be dangerous as these scientists note in a July 16, 2018 news item on phys.org,

Scientists at the Wellcome Sanger Institute have discovered that CRISPR/Cas9 gene editing can cause greater genetic damage in cells than was previously thought. These results create safety implications for gene therapies using CRISPR/Cas9 in the future as the unexpected damage could lead to dangerous changes in some cells.

Reported today (16 July 2018) in the journal Nature Biotechnology, the study also revealed that standard tests for detecting DNA changes miss finding this genetic damage, and that caution and specific testing will be required for any potential gene therapies.

This CRISPR-Cas9 image reminds me of popcorn,

CRISPR-associated protein Cas9 (white) from Staphylococcus aureus based on Protein Database ID 5AXW. Credit: Thomas Splettstoesser (Wikipedia, CC BY-SA 4.0)[ downloaded from https://phys.org/news/2018-07-genome-crisprcas9-gene-higher-thought.html#jCp]

A July 16, 2018 Wellcome Sanger Institute press release (also on EurekAlert), which originated the news item, offers a little more explanation,

CRISPR/Cas9 is one of the newest genome editing tools. It can alter sections of DNA in cells by cutting at specific points and introducing changes at that location. Already extensively used in scientific research, CRISPR/Cas9 has also been seen as a promising way to create potential genome editing treatments for diseases such as HIV, cancer or sickle cell disease. Such therapeutics could inactivate a disease-causing gene, or correct a genetic mutation. However, any potential treatments would have to prove that they were safe.

Previous research had not shown many unforeseen mutations from CRISPR/Cas9 in the DNA at the genome editing target site. To investigate this further the researchers carried out a full systematic study in both mouse and human cells and discovered that CRISPR/Cas9 frequently caused extensive mutations, but at a greater distance from the target site.

The researchers found many of the cells had large genetic rearrangements such as DNA deletions and insertions. These could lead to important genes being switched on or off, which could have major implications for CRISPR/Cas9 use in therapies. In addition, some of these changes were too far away from the target site to be seen with standard genotyping methods.

Prof Allan Bradley, corresponding author on the study from the Wellcome Sanger Institute, said: “This is the first systematic assessment of unexpected events resulting from CRISPR/Cas9 editing in therapeutically relevant cells, and we found that changes in the DNA have been seriously underestimated before now. It is important that anyone thinking of using this technology for gene therapy proceeds with caution, and looks very carefully to check for possible harmful effects.”

Michael Kosicki, the first author from the Wellcome Sanger Institute, said: “My initial experiment used CRISPR/Cas9 as a tool to study gene activity, however it became clear that something unexpected was happening. Once we realised the extent of the genetic rearrangements we studied it systematically, looking at different genes and different therapeutically relevant cell lines, and showed that the CRISPR/Cas9 effects held true.”

The work has implications for how CRISPR/Cas9 is used therapeutically and is likely to re-spark researchers’ interest in finding alternatives to the standard CRISPR/Cas9 method for gene editing.

Prof Maria Jasin, an independent researcher from Memorial Slone Kettering Cancer Centre, New York, who was not involved in the study said: “This study is the first to assess the repertoire of genomic damage arising at a CRISPR/Cas9 cleavage site. While it is not known if genomic sites in other cell lines will be affected in the same way, this study shows that further research and specific testing is needed before CRISPR/Cas9 is used clinically.”

For anyone who’d like to better understand the terms gene editing and CRISPR-Cas9, the Wellcome Sanger Institute provides these explanatory webpages, What is genome editing? and What is CRISPR-Cas9?

For the more advanced, here’s a link and a citation for the paper,

Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements by Michael Kosicki, Kärt Tomberg, & Allan Bradley. Nature Biotechnology DOI: https://doi.org/10.1038/nbt.4192 Published 16 July 2018

This paper appears to be open access.

The kerfuffle

It seems this news has affected the CRISPR market. From a July 16, 2018 article by Cale Guthrie Weissman for Fast Company,

… CRISPR could unknowingly delete or alter non-targeted genes, which could lead to myriad unintended consequences. This is especially frightening, since the technology is going to be used in human clinical trials.

Meanwhile, other scientists working with CRISPR are trying to downplay the findings, telling STAT [a life sciences and business journalism website] that there have been no reported adverse effects similar to what the study describes. The news, however, has brought about a market reaction–at least three publicly traded companies that focus on CRISPR-based therapies are in stock nosedive. Crispr Therapeutics is down by over 6%; Editas fell by over 3%; and Intellia Therapeutics dropped by over 5%. [emphasis mine]

Damage control

Gaetan Burgio (geneticist, Australian National University)  in a July 16, 2018 essay on phys.org (originating from The Conversation) suggests some calm (Note: Links have been removed),

But a new study has called into question the precision of the technique [CRISPR gene editing technology].

The hope for gene editing is that it will be able to cure and correct diseases. To date, many successes have been reported, including curing deafness in mice, and in altering cells to cure cancer.

Some 17 clinical trials in human patients are registered [emphasis mine] testing gene editing on leukaemias, brain cancers and sickle cell anaemia (where red blood cells are misshaped, causing them to die). Before implementing CRISPR technology in clinics to treat cancer or congenital disorders, we must address whether the technique is safe and accurate.

There are a few options for getting around this problem. One option is to isolate the cells we wish to edit from the body and reinject only the ones we know have been correctly edited.

For example, lymphocytes (white blood cells) that are crucial to killing cancer cells could be taken out of the body, then modified using CRISPR to heighten their cancer-killing properties. The DNA of these cells could be sequenced in detail, and only the cells accurately and specifically gene-modified would be selected and delivered back into the body to kill the cancer cells.

While this strategy is valid for cells we can isolate from the body, some cells, such as neurons and muscles, cannot be removed from the body. These types of cells might not be suitable for gene editing using Cas9 scissors.

Fortunately, researchers have discovered other forms of CRISPR systems that don’t require the DNA to be cut. Some CRISPR systems only cut the RNA, not the DNA (DNA contains genetic instructions, RNA convey the instructions on how to synthesise proteins).

As RNA [ribonucleic acid] remains in our cells only for a specific period of time before being degraded, this would allow us to control the timing and duration of the CRISPR system delivery and reverse it (so the scissors are only functional for a short period of time).

This was found to be successful for dementia in mice. Similarly, some CRISPR systems simply change the letters of the DNA, rather than cutting them. This was successful for specific mutations causing diseases such as hereditary deafness in mice.

I agree with Burgio’s conclusion (not included here) that we have a lot more to learn and I can’t help wondering why there are 17 registered human clinical trials at this point.

Nanoparticle-based delivery platform for CRISPR-Cas9 (gene-editing technology)

A February 18, 2018 King Abdullah University of Science and Technology (KAUST; Saudi Arabia) news release (also on EurekAlert but published on Feb. 20, 2018) describes a new technology for delivering CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 into cells,

A new delivery system for introducing gene-editing technology into cells could help safely and efficiently correct disease-causing mutations in patients.

The system, developed by KAUST scientists, is the first to use sponge-like ensembles of metal ions and organic molecules to coat the molecular components of the precision DNA-editing technology known as CRISPR/Cas9, allowing efficient release of the genome-editing machinery inside the cell.

“This method presents an easy and economically feasible route to improve on the delivery problems that accompany RNA-based therapeutic approaches,” says Niveen Khashab, the associate professor of chemical sciences at KAUST who led the study. “This may permit such formulations to be eventually used for treating genetic diseases effectively in the future.”

CRISPR/Cas9 has a double delivery problem: For the gene-editing technology to work like a molecular Swiss Army knife, both a large protein (the Cas9 cutting enzyme) and a highly charged RNA component (the guide RNA used for DNA targeting) must each get from the outside of the cell into the cytoplasm and finally into the nucleus, all without getting trapped in the tiny intracellular bubbles that are known as endosomes.

To solve this problem, Khashab and her lab turned to a nano-sized type of porous material known as a zeolitic imidazolate framework, which forms a cage-like structure into which other molecules can be placed. The researchers encapsulated the Cas9 protein and guide RNA in this material and then introduced the resulting nanoparticles into hamster cells.

The encapsulated CRISPR-Cas9 constructs were not toxic to the cells. And because particles in the coating material become positively charged when absorbed into endosomes, they caused these membrane-bound bubbles to burst, freeing the CRISPR-Cas9 machinery to travel to the nucleus, home to the cell’s genome. There the gene-editing technology could get to work.

Using a guide RNA designed to target a gene that caused the cells to glow green under fluorescent light, Khashab and her team showed that they could reduce the expression of this gene by 37 percent over four days with their technology. “These cage-like structures are biocompatible and can be triggered on demand, making them smart options to overcome delivery problems of genetic materials and proteins,” says the study’s first author Shahad Alsaiari, a Ph.D. student in Khashab’s lab.

The researchers’ plan to test their system in human cells and in mice, and eventually, they hope, in clinical trials.

The zeolitic imidazolate framework forms a cage-like scaffold over the CRISPR/Cas9 machinery.. Reprinted (adapted) with permission from Alsaiari, S.K., Patil, S., Alyami, M., Alamoudi, K.O., Aleisa, F.A., Merzaban, J., Li M. & Khashab, N.M. Endosomal escape and delivery of CRISPR/Cas9 genome editing machinery enabled by nanoscale zeolitic imidazolate framework. Journal of the American Chemical Society 140, 143–146 (2018). © 2018 American Chemical Society; KAUST Xavier Pita and Heno Huang ][downloaded from https://discovery.kaust.edu.sa/en/article/475/a%250adelivery-platform-for-gene-editing-technology]

Here’s a link to and a citation for the paper,

Endosomal Escape and Delivery of CRISPR/Cas9 Genome Editing Machinery Enabled by Nanoscale Zeolitic Imidazolate Framework by Shahad K. Alsaiari, Sachin Patil, Mram Alyami, Kholod O. Alamoudi, Fajr A. Aleisa, Jasmeen S. Merzaban, Mo Li, and Niveen M. Khashab. J. Am. Chem. Soc., 2018, 140 (1), pp 143–146 DOI: 10.1021/jacs.7b11754 Publication Date (Web): December 22, 2017

Copyright © 2017 American Chemical Society

This paper is behind a paywall.

CRISPR-Cas12a as a new diagnostic tool

Similar to Cas9, Cas12a is has an added feature as noted in this February 15, 2018 news item on ScienceDaily,

Utilizing an unsuspected activity of the CRISPR-Cas12a protein, researchers created a simple diagnostic system called DETECTR to analyze cells, blood, saliva, urine and stool to detect genetic mutations, cancer and antibiotic resistance and also diagnose bacterial and viral infections. The scientists discovered that when Cas12a binds its double-stranded DNA target, it indiscriminately chews up all single-stranded DNA. They then created reporter molecules attached to single-stranded DNA to signal when Cas12a finds its target.

A February 15, 2018 University of California at Berkeley (UC Berkeley) news release by Robert Sanders and which originated the news item, provides more detail and history,

CRISPR-Cas12a, one of the DNA-cutting proteins revolutionizing biology today, has an unexpected side effect that makes it an ideal enzyme for simple, rapid and accurate disease diagnostics.

blood in test tube

(iStock)

Cas12a, discovered in 2015 and originally called Cpf1, is like the well-known Cas9 protein that UC Berkeley’s Jennifer Doudna and colleague Emmanuelle Charpentier turned into a powerful gene-editing tool in 2012.

CRISPR-Cas9 has supercharged biological research in a mere six years, speeding up exploration of the causes of disease and sparking many potential new therapies. Cas12a was a major addition to the gene-cutting toolbox, able to cut double-stranded DNA at places that Cas9 can’t, and, because it leaves ragged edges, perhaps easier to use when inserting a new gene at the DNA cut.

But co-first authors Janice Chen, Enbo Ma and Lucas Harrington in Doudna’s lab discovered that when Cas12a binds and cuts a targeted double-stranded DNA sequence, it unexpectedly unleashes indiscriminate cutting of all single-stranded DNA in a test tube.

Most of the DNA in a cell is in the form of a double-stranded helix, so this is not necessarily a problem for gene-editing applications. But it does allow researchers to use a single-stranded “reporter” molecule with the CRISPR-Cas12a protein, which produces an unambiguous fluorescent signal when Cas12a has found its target.

“We continue to be fascinated by the functions of bacterial CRISPR systems and how mechanistic understanding leads to opportunities for new technologies,” said Doudna, a professor of molecular and cell biology and of chemistry and a Howard Hughes Medical Institute investigator.

DETECTR diagnostics

The new DETECTR system based on CRISPR-Cas12a can analyze cells, blood, saliva, urine and stool to detect genetic mutations, cancer and antibiotic resistance as well as diagnose bacterial and viral infections. Target DNA is amplified by RPA to make it easier for Cas12a to find it and bind, unleashing indiscriminate cutting of single-stranded DNA, including DNA attached to a fluorescent marker (gold star) that tells researchers that Cas12a has found its target.

The UC Berkeley researchers, along with their colleagues at UC San Francisco, will publish their findings Feb. 15 [2018] via the journal Science’s fast-track service, First Release.

The researchers developed a diagnostic system they dubbed the DNA Endonuclease Targeted CRISPR Trans Reporter, or DETECTR, for quick and easy point-of-care detection of even small amounts of DNA in clinical samples. It involves adding all reagents in a single reaction: CRISPR-Cas12a and its RNA targeting sequence (guide RNA), fluorescent reporter molecule and an isothermal amplification system called recombinase polymerase amplification (RPA), which is similar to polymerase chain reaction (PCR). When warmed to body temperature, RPA rapidly multiplies the number of copies of the target DNA, boosting the chances Cas12a will find one of them, bind and unleash single-strand DNA cutting, resulting in a fluorescent readout.

The UC Berkeley researchers tested this strategy using patient samples containing human papilloma virus (HPV), in collaboration with Joel Palefsky’s lab at UC San Francisco. Using DETECTR, they were able to demonstrate accurate detection of the “high-risk” HPV types 16 and 18 in samples infected with many different HPV types.

“This protein works as a robust tool to detect DNA from a variety of sources,” Chen said. “We want to push the limits of the technology, which is potentially applicable in any point-of-care diagnostic situation where there is a DNA component, including cancer and infectious disease.”

The indiscriminate cutting of all single-stranded DNA, which the researchers discovered holds true for all related Cas12 molecules, but not Cas9, may have unwanted effects in genome editing applications, but more research is needed on this topic, Chen said. During the transcription of genes, for example, the cell briefly creates single strands of DNA that could accidentally be cut by Cas12a.

The activity of the Cas12 proteins is similar to that of another family of CRISPR enzymes, Cas13a, which chew up RNA after binding to a target RNA sequence. Various teams, including Doudna’s, are developing diagnostic tests using Cas13a that could, for example, detect the RNA genome of HIV.

infographic about DETECTR system

(Infographic by the Howard Hughes Medical Institute)

These new tools have been repurposed from their original role in microbes where they serve as adaptive immune systems to fend off viral infections. In these bacteria, Cas proteins store records of past infections and use these “memories” to identify harmful DNA during infections. Cas12a, the protein used in this study, then cuts the invading DNA, saving the bacteria from being taken over by the virus.

The chance discovery of Cas12a’s unusual behavior highlights the importance of basic research, Chen said, since it came from a basic curiosity about the mechanism Cas12a uses to cleave double-stranded DNA.

“It’s cool that, by going after the question of the cleavage mechanism of this protein, we uncovered what we think is a very powerful technology useful in an array of applications,” Chen said.

Here’s a link to and a citation for the paper,

CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity by Janice S. Chen, Enbo Ma, Lucas B. Harrington, Maria Da Costa, Xinran Tian, Joel M. Palefsky, Jennifer A. Doudna. Science 15 Feb 2018: eaar6245 DOI: 10.1126/science.aar6245

This paper is behind a paywall.

Stretchy optical materials for implants that could pulse light

An Oct. 17, 2016 Massachusetts Institute of Technology (MIT) news release (also on EurekAlert) by Emily Chu describes research that could lead to long-lasting implants offering preventive health strategies,

Researchers from MIT and Harvard Medical School have developed a biocompatible and highly stretchable optical fiber made from hydrogel — an elastic, rubbery material composed mostly of water. The fiber, which is as bendable as a rope of licorice, may one day be implanted in the body to deliver therapeutic pulses of light or light up at the first sign of disease. [emphasis mine]

The researchers say the fiber may serve as a long-lasting implant that would bend and twist with the body without breaking down. The team has published its results online in the journal Advanced Materials.

Using light to activate cells, and particularly neurons in the brain, is a highly active field known as optogenetics, in which researchers deliver short pulses of light to targeted tissues using needle-like fibers, through which they shine light from an LED source.

“But the brain is like a bowl of Jell-O, whereas these fibers are like glass — very rigid, which can possibly damage brain tissues,” says Xuanhe Zhao, the Robert N. Noyce Career Development Associate Professor in MIT’s Department of Mechanical Engineering. “If these fibers could match the flexibility and softness of the brain, they could provide long-term more effective stimulation and therapy.”

Getting to the core of it

Zhao’s group at MIT, including graduate students Xinyue Liu and Hyunwoo Yuk, specializes in tuning the mechanical properties of hydrogels. The researchers have devised multiple recipes for making tough yet pliable hydrogels out of various biopolymers. The team has also come up with ways to bond hydrogels with various surfaces such as metallic sensors and LEDs, to create stretchable electronics.

The researchers only thought to explore hydrogel’s use in optical fibers after conversations with the bio-optics group at Harvard Medical School, led by Associate Professor Seok-Hyun (Andy) Yun. Yun’s group had previously fabricated an optical fiber from hydrogel material that successfully transmitted light through the fiber. However, the material broke apart when bent or slightly stretched. Zhao’s hydrogels, in contrast, could stretch and bend like taffy. The two groups joined efforts and looked for ways to incorporate Zhao’s hydrogel into Yun’s optical fiber design.

Yun’s design consists of a core material encased in an outer cladding. To transmit the maximum amount of light through the core of the fiber, the core and the cladding should be made of materials with very different refractive indices, or degrees to which they can bend light.

“If these two things are too similar, whatever light source flows through the fiber will just fade away,” Yuk explains. “In optical fibers, people want to have a much higher refractive index in the core, versus cladding, so that when light goes through the core, it bounces off the interface of the cladding and stays within the core.”

Happily, they found that Zhao’s hydrogel material was highly transparent and possessed a refractive index that was ideal as a core material. But when they tried to coat the hydrogel with a cladding polymer solution, the two materials tended to peel apart when the fiber was stretched or bent.

To bond the two materials together, the researchers added conjugation chemicals to the cladding solution, which, when coated over the hydrogel core, generated chemical links between the outer surfaces of both materials.

“It clicks together the carboxyl groups in the cladding, and the amine groups in the core material, like molecular-level glue,” Yuk says.

Sensing strain

The researchers tested the optical fibers’ ability to propagate light by shining a laser through fibers of various lengths. Each fiber transmitted light without significant attenuation, or fading. They also found that fibers could be stretched over seven times their original length without breaking.

Now that they had developed a highly flexible and robust optical fiber, made from a hydrogel material that was also biocompatible, the researchers began to play with the fiber’s optical properties, to see if they could design a fiber that could sense when and where it was being stretched.

They first loaded a fiber with red, green, and blue organic dyes, placed at specific spots along the fiber’s length. Next, they shone a laser through the fiber and stretched, for instance, the red region. They measured the spectrum of light that made it all the way through the fiber, and noted the intensity of the red light. They reasoned that this intensity relates directly to the amount of light absorbed by the red dye, as a result of that region being stretched.

In other words, by measuring the amount of light at the far end of the fiber, the researchers can quantitatively determine where and by how much a fiber was stretched.

“When you stretch a certain portion of the fiber, the dimensions of that part of the fiber changes, along with the amount of light that region absorbs and scatters, so in this way, the fiber can serve as a sensor of strain,” Liu explains.

“This is like a multistrain sensor through a single fiber,” Yuk adds. “So it can be an implantable or wearable strain gauge.”

The researchers imagine that such stretchable, strain-sensing optical fibers could be implanted or fitted along the length of a patient’s arm or leg, to monitor for signs of improving mobility.

Zhao envisions the fibers may also serve as sensors, lighting up in response to signs of disease.

“We may be able to use optical fibers for long-term diagnostics, to optically monitor tumors or inflammation,” he says. “The applications can be impactful.”

Here’s a link to and a citation for the paper,

Highly Stretchable, Strain Sensing Hydrogel Optical Fibers by Jingjing Guo, Xinyue Liu, Nan Jiang, Ali K. Yetisen, Hyunwoo Yuk, Changxi Yang, Ali Khademhosseini, Xuanhe Zhao, and Seok-Hyun Yun. Advanced Materials DOI: 10.1002/adma.201603160 Version of Record online: 7 OCT 2016

© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

This paper is behind a paywall.

Watching artificial nanofibres self-sort in real-time

A May 31, 2016 news item on phys.org describes research on self-assembling fibres at Kyoto University (Japan) by referencing the ancient Greek mythological figure, Psyche,

The Greek goddess Psyche borrowed help from ants to sort a room full of different grains. Cells, on the other hand, do something similar without Olympian assistance, as they organize molecules into robust, functional fibers. Now scientists are able to see self-sorting phenomena happen in real time with artificial molecules.

The achievement, reported in Nature Chemistry, elucidates how two different types of nanofibers sort themselves into organized structures under artificial conditions.

“Basic cellular structures, such as actin filaments, come into being through the autonomous self-sorting of individual molecules, even though a tremendous variety of proteins and small molecules are present inside the cell,” says lead author Hajime Shigemitsu, a researcher in Itaru Hamachi’s lab at Kyoto University.

A May 30, 2016 Kyoto University news release (also on EurekAlert), which originated the news item, expands on the theme,

“Imagine a box filled with an assortment of building blocks — it’s as if the same type of blocks started sorting themselves into neat bundles all on their own. In living cells, such phenomena always happen, enabling accurate self-assembling of proteins, which is essential for cell functions.”

“If we are able to control self-sorting with artificial molecules, we can work toward developing intelligent, next-generation biomimics that possess the flexibility and diversity of functions that exist in a living cell.”

Study co-author Ryou Kubota explains that previous studies have already made artificial molecules build themselves into fibers — but only when there was one type of molecule around. Having a jumble of types, on the other hand, made the molecules confused.

“The difficulty in inducing self-assembly with artificial molecules is that they don’t recognize the same type of molecule, unlike molecules in the natural world. Different types of artificial molecules interact with each other and make an unsorted cluster.”

From a database of structural analyses, Hamachi and colleagues discovered a combination of nanofibers — namely a peptide-based and lipid-based hydrogelator — that would make sorted fibers without mixing with the other. They then tethered the fibers with fluorescent probes; with a type of microscope typically used in cell imaging, the team was able to observe directly and in real-time how the artificial molecules sorted themselves.

“Ultimately, this finding could help develop new materials that respond dynamically to different environments and stimuli,” elaborates Hamachi. “This insight is not only useful for materials science, but may also provide useful clues for understanding self-organization in cells.”

Here’s a link to and a citation for the paper,

In situ real-time imaging of self-sorted supramolecular nanofibres by Shoji Onogi, Hajime Shigemitsu, Tatsuyuki Yoshii, Tatsuya Tanida, Masato Ikeda, Ryou Kubota, & Itaru Hamachi. Nature Chemistry (2016) doi:10.1038/nchem.2526 Published online 30 May 2016

This paper is behind a paywall bu the researchers have made a video of the self-sorting proteins freely available,

Cutting into a cell with a nanoblade

A May 11, 2016 news item on Nanotechnology Now features a type of surgery that could aid in cell engineering,

To study certain aspects of cells, researchers need the ability to take the innards out, manipulate them, and put them back. Options for this kind of work are limited, but researchers reporting May 10 [2016] in Cell Metabolism describe a “nanoblade” that can slice through a cell’s membrane to insert mitochondria. The researchers have previously used this technology to transfer other materials between cells and hope to commercialize the nanoblade for wider use in bioengineering.

Caption: This diagram illustrates the process of transferring mitochondria between cells using the nanoblade technology. Credit: Alexander N. Patananan Courtesy UCLA

Caption: This diagram illustrates the process of transferring mitochondria between cells using the nanoblade technology.
Credit: Alexander N. Patananan Courtesy UCLA

A May 10, 2016 Cell Press news release on EurekAlert, which originated the news item, expands on the theme,

“As a new tool for cell engineering, to truly engineer cells for health purposes and research, I think this is very unique,” says Mike Teitell, a pathologist and bioengineer at the University of California, Los Angeles (UCLA). “We haven’t run into anything so far, up to a few microns in size, that we can’t deliver.”

Teitell and Pei-Yu “Eric” Chiou, also a bioengineer at UCLA, first conceived the idea of a nanoblade several years ago to transfer a nucleus from one cell to another. However, they soon delved into the intersection of stem cell biology and energy metabolism, where the technology could be used to manipulate a cell’s mitochondria. Studying the effects of mutations in the mitochondrial genome, which can cause debilitating or fatal diseases in humans, is tricky for a number of reasons.

“There’s a bottleneck in the field for modifying a cell’s mitochondrial DNA,” says Teitell. “So we are working on a two-step process: edit the mitochondrial genome outside of a cell, and then take those manipulated mitochondria and put them back into the cell. We’re still working on the first step, but we’ve solved that second one quite well.”

The nanoblade apparatus consists of a microscope, laser, and titanium-coated micropipette to act as the “blade,” operated using a joystick controller. When a laser pulse strikes the titanium, the metal heats up, vaporizing the surrounding water layers in the culture media and forming a bubble next to a cell. Within a microsecond, the bubble expands, generating a local force that punctures the cell membrane and creates a passageway several microns long that the “cargo”–in this case, mitochondria–can be pushed through. The cell then rapidly repairs the membrane defect.

Teitell, Chiou, and their team used the nanoblade to insert tagged mitochondria from human breast cancer cells and embryonic kidney cells into cells without mitochondrial DNA. When they sequenced the nuclear and mitochondrial DNA afterwards, the researchers saw that the mitochondria had been successfully transferred and replicated by 2% of the cells, with a range of functionality. Other methods of mitochondrial transfer are hard to control, and when they have been reported to work, the success rates have been only 0.0001%-0.5% according to the researchers.

“The success of the mitochondrial transfer was very encouraging,” says Chiou. “The most exciting application for the nanoblade, to me, is in the study of mitochondria and infectious diseases. This technology brings new capabilities to help advance these fields.”

The team’s aspirations also go well beyond mitochondria, and they’ve already scaled up the nanoblade apparatus into an automated high-throughput version. “We want to make a platform that’s easy to use for everyone and allow researchers to devise anything they can think of a few microns or smaller that would be helpful for their research–whether that’s inserting antibodies, pathogens, synthetic materials, or something else that we haven’t imagined,” says Teitell. “It would be very cool to allow people to do something that they can’t do right now.”

The pipette being used is measured at the microscale but it’s called a nanoblade? Well, perhaps the tip or the edge of the pipette is measured at the nanoscale.

Getting back to the research, here’s a link to and a citation for the paper,

Mitochondrial Transfer by Photothermal Nanoblade Restores Metabolite Profile in Mammalian Cells by Ting-Hsiang Wu, Enrico Sagullo, Dana Case, Xin Zheng, Yanjing Li, Jason S. Hong, Tara TeSlaa, Alexander N. Patananan, J. Michael McCaffery, Kayvan Niazi, Daniel Braas, Carla M. Koehler, Thomas G. Graeber, Pei-Yu Chiou, Michael A. Teitell. Cell Metabolism Volume 23, Issue 5, p921–929, 10 May 2016  DOI: http://dx.doi.org/10.1016/j.cmet.2016.04.007

This paper appears to be open access.

A nanoscale bacteria power grid

It’s not often you see the word ‘spectacular’ when reading a science news item but it can be found in an Oct. 21, 2015 news item on ScienceDaily,

Electrical energy from the socket — this convenient type of power supply is apparently used by some microorganisms. Cells can meet their energy needs in the form of electricity through nano-wire connections. Researchers from the Max Planck Institute for Marine Microbiology in Bremen have discovered these possibly smallest power grids in the world when examining cell aggregates of methane degrading microorganisms. They consist of two completely different cell types, which can only jointly degrade methane. Scientists have discovered wire-like connections between the cells, which are relevant in energy exchanges.

It was a spectacular [emphasis mine] scientific finding when researchers discovered electrical wiring between microorganisms using iron as energy source in 2010. Immediately the question came up if electric power exchange is common in other microbially mediated reactions. One of the processes in question was the anaerobic oxidation of methane (AOM) that is responsible for the degradation of the greenhouse gas methane in the seafloor, and therefore has a great relevance for Earth climate. The microorganisms involved have been described for the first time in 2000 by researchers from Bremen and since then have been extensively studied.

This image accompanies the research,

Caption: Electron micrograph of the nanowires shows connecting archaea and sulphate reducing bacteria. The wires stretch out for several micrometres, longer than a single cell. The white bar represents the length of one micrometre. The arrows indicate the nanowires (A=ANME-Archaeen, H=HotSeep-1 partner bacteria). Credit: MPI f. Biophysical Chemistry

Caption: Electron micrograph of the nanowires shows connecting archaea and sulphate reducing bacteria. The wires stretch out for several micrometres, longer than a single cell. The white bar represents the length of one micrometre. The arrows indicate the nanowires (A=ANME-Archaeen, H=HotSeep-1 partner bacteria).
Credit: MPI f. Biophysical Chemistry

A Oct. 21, 2015 Max Planck press release (also on EurekAlert), which originated the news item, provides more information about methane in the ocean, power wires, and electron transporters,

In the ocean, methane is produced from the decay of dead biomass in subsurface sediments. The methane rises upwards to the seafloor, but before reaching the water column it is degraded by special consortia of archaea and bacteria. The archaea take up methane and oxidise it to carbonate. They pass on energy to their partner bacteria, so that the reaction can proceed. The bacteria respire sulphate instead of oxygen to gain energy (sulphate reducers). This may be an ancient metabolism, already relevant billions of years ago when the Earth’s atmosphere was oxygen-free. Yet today it remains unknown how the anaerobic oxidation of methane works biochemically.

Gunter Wegener, who authors the publication together with PhD student Viola Krukenberg, says: “We focused on thermophilic AOM consortia living at 60 degrees Celsius. For the first time we were able to isolate the partner bacteria to grow them alone. Then we systematically compared the physiology of the isolate with that of the AOM culture. We wanted to know which substances can serve as an energy carrier between the archaea and sulphate reducers.” Most compounds were ruled out quickly. At first, hydrogen was considered as energy source. However, the archaea did not produce sufficient hydrogen to explain the growth of sulphate reducers – hence the researchers had to change their strategy.

Direct power wires and electron transporters

One possible alternative was to look for direct connections channelling electrons between the cells. Using electron microscopy on the thermophilic AOM cultures this idea was confirmed. Dietmar Riedel, head of electron microscopy facilities at the Max Planck Institute in Goettingen says: “It was really challenging to visualize the cable-like structures. We embedded aggregates under high pressure using different embedding media. Ultrathin sections of these aggregates were then examined in near-native state using transmission electron microscopy.”

Viola Krukenberg adds: “We found all genes necessary for biosynthesis of the cellular connections called pili. Only when methane is added as energy source these genes are activated and pili are formed between bacteria and archaea.”

With length of several micrometres the wires can exceed the length of the cells by far, but their diameter is only a few nanometres. These wires provide the contact between the closely spaced cells and explain the spatial structure of the consortium, as was shown by a team of researchers led by Victoria Orphan from Caltech.

“Consortia of archaea and bacteria are abundant in nature. Our next step is to see whether other types also show such nanowire-like connections. It is important to understand how methane-degrading microbial consortia work, as they provide important functions in nature”, explains Antje Boetius, leader of the research group at the Institute in Bremen.

Here’s a link to and a citation for the paper,

Intercellular wiring enables electron transfer between methanotrophic archaea and bacteria by Gunter Wegener, Viola Krukenberg, Dietmar Riedel, Halina E. Tegetmeyer, & Antje Boetius. Nature 526, 587–590 (22 October 2015) doi:10.1038/nature15733 Published online 21 October 2015

This paper is behind a paywall.

Hitchhikers at the nanoscale show how cells stir themselves

A May 30, 2014 news item on Nanowerk highlights some molecule-tracking research,

Chemical engineers from Rice University and biophysicists from Georg-August Universität Göttingen in Germany and the VU University Amsterdam in the Netherlands have successfully tracked single molecules inside living cells with carbon nanotubes.

Through this new method, the researchers found that cells stir their interiors using the same motor proteins that serve in muscle contraction.

A May 29, 2014 Rice University news release by Mike Williams, which originated the news item, describes the researchers’ work,

The team attached carbon nanotubes to transport molecules known as kinesin motors to visualize and track them as they moved through the cytoplasm of living cells.

Carbon nanotubes are hollow cylinders of pure carbon with one-atom-thick walls. They naturally fluoresce with near-infrared wavelengths when exposed to visible light, a property discovered at Rice by Professor Rick Smalley a decade ago and then leveraged by Rice Professor Bruce Weisman to image carbon nanotubes. When attached to a molecule, the hitchhiking nanotubes serve as tiny beacons that can be precisely tracked over long periods of time to investigate small, random motions inside cells.

“Any probe that can hitch the length and breadth of the cell, rough it, slum it, struggle against terrible odds, win through and still know where its protein is, is clearly a probe to be reckoned with,” said lead author Nikta Fakhri, paraphrasing “The Hitchhiker’s Guide to the Galaxy.” Fakhri, who earned her Rice doctorate in Pasquali’s lab in 2011, is currently a Human Frontier Science Program Fellow at Göttingen.

“In fact, the exceptional stability of these probes made it possible to observe intracellular motions from times as short as milliseconds to as long as hours,” she said.

For long-distance transport, such as along the long axons of nerve cells, cells usually employ motor proteins tied to lipid vesicles, the cell’s “cargo containers.” This process involves considerable logistics: Cargo needs to be packed, attached to the motors and sent off in the right direction.

“This research has helped uncover an additional, much simpler mechanism for transport within the cell interior,” said principal investigator Christoph Schmidt, a professor of physics at Göttingen. “Cells vigorously stir themselves, much in the way a chemist would accelerate a reaction by shaking a test tube. This will help them to move objects around in the highly crowded cellular environment.”

The researchers showed the same type of motor protein used for muscle contraction is responsible for stirring. They reached this conclusion after exposing the cells to drugs that suppressed these specific motor proteins. The tests showed that the stirring was suppressed as well.

The mechanical cytoskeleton of cells consists of networks of protein filaments, like actin. Within the cell, the motor protein myosin forms bundles that actively contract the actin network for short periods. The researchers found random pinching of the elastic actin network by many myosin bundles resulted in the global internal stirring of the cell. Both actin and myosin play a similar role in muscle contraction.

The highly accurate measurements of internal fluctuations in the cells were explained in a theoretical model developed by VU co-author Fred MacKintosh, who used the elastic properties of the cytoskeleton and the force-generation characteristics of the motors.

“The new discovery not only promotes our understanding of cell dynamics, but also points to interesting possibilities in designing ‘active’ technical materials,” said Fakhri, who will soon join the Massachusetts Institute of Technology faculty as an assistant professor of physics. “Imagine a microscopic biomedical device that mixes tiny samples of blood with reagents to detect disease or smart filters that separate squishy from rigid materials.”

There is an accompanying video,

This video is typical of the kind of visual image that nanoscientists look at and provides an interesting contrast to ‘nano art’ where colours and other enhancements are added. as per this example, NanoOrchard, from a May 13, 2014 news item on Nanowerk about the 2014 Materials Research Society spring meeting and their Science as Art competition,

NanoOrchard – Electrochemically overgrown CuNi nanopillars. (Image courtesy of the Materials Research Society Science as Art Competition and Josep Nogues, Institut Catala de Nanociencia i Nanotecnologia (ICN2), Spain, and A. Varea, E. Pellicer, S. Suriñach, M.D. Baro, J. Sort, Univ. Autonoma de Barcelona) [downloaded from http://www.nanowerk.com/nanotechnology-news/newsid=35631.php]

NanoOrchard – Electrochemically overgrown CuNi nanopillars. (Image courtesy of the Materials Research Society Science as Art Competition and Josep Nogues, Institut Catala de Nanociencia i Nanotecnologia (ICN2), Spain, and A. Varea, E. Pellicer, S. Suriñach, M.D. Baro, J. Sort, Univ. Autonoma de Barcelona) [downloaded from http://www.nanowerk.com/nanotechnology-news/newsid=35631.php]

Getting back to the carbon nanotube hitchhikers, here’s a link to and a citation for the paper,

High-resolution mapping of intracellular fluctuations using carbon nanotubes by Nikta Fakhri, Alok D. Wessel, Charlotte Willms, Matteo Pasquali, Dieter R. Klopfenstein, Frederick C. MacKintosh, and Christoph F. Schmidt. Science 30 May 2014: Vol. 344 no. 6187 pp. 1031-1035 DOI: 10.1126/science.1250170

This article is behind a paywall.

One final comment, I am delighted by the researcher’s reference to the Hitchhiker’s Guide to the Galaxy.

Hands, Waldo, and nano-scalpels

Hands were featured in Waldo (a 1943 short story by Robert Heinlein) and in Richard Feynman’s “Plenty of room at the bottom” 1959 lecture both of which were concerned with describing a field we now call nanotechnology. As I put it in my Aug. 17, 2009 posting,

Both of these texts feature the development of ‘smaller and smaller robotic hands to manipulate matter at the atomic and molecular levels’ and both of these have been cited as the birth of nanotechnology.

The details are a bit sketchy but it seems that scientists at the University of Bath (UK) have created a tiny (nanoscale) tool that looks like a hand. From the University of Bath’s Dec. 12, 2011 news release,

The lower picture shows the AFM probe with the nano-hand circled. The upper image is a vastly enlarged image of the nano-hand, showing the beckoning motion spotted by Dr Gordeev.

Here’s a little more about Dr. Gordeev’s observation from the Dec. 12, 2011 news item on Nanowerk,

Dr Sergey Gordeev, from the Department of Physics, was trying to create a nano-scalpel, a tool which can be used by biologists to look inside cells, when the process went wrong.

Dr Gordeev said: “I was amazed when I looked at the nano-scalpel and saw what appeared to be a beckoning hand.

“Nanoscience research is moving very fast at the moment, so maybe the nano-hand is trying to attract people and funders into this area.

The research group is using funding from Bath Ventures, an organisation which commercialises the results of the University’s research, and private company Diamond Hard Surfaces Ltd, to explore the use of hard coatings for nano-tools, making them more durable and suitable for delicate biological procedures.

I appreciate Dr. Gordeev’s whimsical notion that the hand might be trying to attract funding for this research group.

Cells and transistors

Analog/digital, is there a difference? After reading the latest from MIT’s (Massachusetts Institute of Technology) Research Laboratory Electronics (RLE), the answer turns out to be no, when it comes to transistors. From the Sept. 29, 2011 news item on Nanowerk,

A transistor is basically a switch: When it’s on, it conducts electricity; when it’s off, it doesn’t. [emphases mine] In a computer chip, those two states represent 0s and 1s.

But in moving between its nonconductive and conductive states, a transistor passes through every state in between — slightly conductive, moderately conductive, fairly conductive — just as a car accelerating from zero to 60 passes through every speed in between. Because the transistors in a computer chip are intended to perform binary logic operations, they’re designed to make those transitional states undetectable. [emphases mine]

The MIT researchers will be discussing their work using analog transistors to increase the concentrations of two different proteins in cells. From the news item on Nanowerk,

At the Biomedical Circuits and Systems Conference in San Diego in November, Sarpeshkar [Rahul Sarpeshkar, associate professor of electrical engineering], research scientist Lorenzo Turicchia, postdoc Ramiz Daniel and graduate student Sung Sik Woo, all of RLE, will present a paper in which they use analog electronic circuits to model two different types of interactions between proteins and DNA in the cell. The circuits mimic the behaviors of the cell with remarkable accuracy, but perhaps more important, they do it with far fewer transistors than a digital model would require.

Here’s a graphic representation of transistors in a cell (downloaded from the MIT News Office page for this research,

Graphic: Christine Daniloff

This works seems to be signaling (pun noted) a change in how systems biology and synthetic biology researchers think about biological systems. From the Sept. 28, 2011 news item by Larry Hardesty for the MIT News Office,

Since the completion of the Human Genome Project, two thriving new disciplines — synthetic biology and systems biology — have emerged from the observation that in some ways, the sequences of chemical reactions that lead to protein production in cells are a lot like electronic circuits. In general, researchers in both fields tend to analyze reactions in terms of binary oppositions: If a chemical is present, one thing happens; if the chemical is absent, a different thing happens.

But Rahul Sarpeshkar, an associate professor of electrical engineering in MIT’s Research Laboratory of Electronics (RLE), thinks that’s the wrong approach. “The signals in cells are not ones or zeroes,” Sarpeshkar says. “That’s an overly simplified abstraction that is kind of a first, crude, useful approximation for what cells do. But everybody knows that’s really wrong.”

From what I understand of the synthetic biology and systems biology communities, this is a major change.