NanoLuc: Tiny Tag with a Big Impact

Synthetic biology—genetically engineering an organism to do or make something useful—is the central goal of the iGEM competition each year. After teams conquer the challenge of cloning their gene, the next hurdle is demonstrating that the engineered gene is expressing the desired protein (and possibly quantifying the level of expression), which they may do using a reporter gene.

Reporters can also play a more significant role in iGEM projects when teams design their organism with reporter genes to detect and signal the presence of specific molecules, like environmental toxins or biomarkers. Three of the iGEM teams Promega sponsored this year opted to incorporate some version of NanoLuc® Luciferase into their projects.

NanoLuc® luciferase is a small monomeric enzyme (19.1kDa, 171 amino acids) based on the luciferase from the deep sea shrimp Oplophorus gracilirostris. This engineered enzyme uses a novel substrate, furimazine, to produce high-intensity, glow-type luminescence in an ATP-independent reaction. Unlike other molecules for tagging and detecting proteins, NanoLuc® luciferase is less likely to interfere with enzyme activity and affect protein production due to its small size.

NanoLuc® Luciferase has also been engineered into a structural complementation reporter system, NanoBiT® Luciferase, that contains a Large subunit (LgBiT) and two small subunit options: low affinity SmBiT and high affinity HiBiT. Together, these NanoLuc® technologies provide a bioluminescent toolbox that was used by the iGEM teams to address a diverse set of biological challenges.

Here is an overview of each team’s project and how they incorporated NanoLuc® technology.

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Out-FOXOing High-Stage Neuroblastoma

Fluorescence microscopy of neuroblastoma cells.

In recent years, scientists have been hot on the trail of transcription factor FOXO3, tracing its involvement in various tumor-centric activities comprising the many trademarks of cancer, from drug resistance to metastasis to tumor angiogenesis.

FOXO3 is a member of the O sub-class of the forkhead box family of transcription factors. The forkhead box (FOX) family is characterized by a fork head DNA-binding domain (DBD), comprised of around 100 amino acids. They have also proven themselves to be a family of many hats, functioning in diverse roles ranging from metabolism, immunology, cell-cycle control, development, as well as cancer (1). The forkhead box O (FOXO) sub-class alone has demonstrated involvement in a variety of cellular outcomes, from drug resistance and longevity to apoptosis induction.

Due to its pro-apoptotic and anti-proliferative proclivity, FOXO3 has been previously identified as a tumor suppressor gene. However, more and more studies have begun to flip the narrative on FOXO3, portraying it more as a devoted henchman, due to its roles in drug and radiotherapy resistance, cell-cycle arrest and long-term maintenance of leukemia-initiating stem cells in a variety of cancer types, including breast cancer, pancreatic cancer, glioblastoma, and both acute and chronic myeloid leukemia.

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MSI Testing of Tumor Cells for Better Tailored Treatment

There are as many different cancers as there are people with cancer. Unlike infectious diseases, which are caused by pathogens that are foreign to our bodies (bacteria, viruses, parasites), cancer cells arise from our body—our own cells gone rogue. Because cancer is a dysfunction of a person’s normal cells, every cancer reflects the genetic differences that mark us as individuals. Add to that environmental influences like diet, tobacco use, the microbiome and even occupation, and the likelihood of finding a “single” pharmaceutical cure for cancer becomes virtually impossible.

But, while looking for a single cure for all cancers may not be a fruitful activity, defining a best practice for understanding the genetic and protein biomarkers of individual tumors is proving worthwhile.

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Discussing the Future of Gene Editing at CRISPRcon Midwest

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Walking in to the first session at CRISPRcon Midwest.

Last week, a diverse group of stakeholders attended CRISPRcon Midwest, hosted by the Keystone Policy Center and the University of Wisconsin–Madison. The goal of the day-long conference was to emphasize the importance and value of gene editing technology, and how it must be communicated deliberately between scientists, the public, policymakers, and other stakeholders.

Julie Shapiro, Senior Policy Director of Keystone Policy Center, acted as Emcee for the event. Given the diverse group of attendees, she mentioned in her opening remarks that the event organizers were “seeking conversation, not consensus” and emphasized the “power of respectful dialogue.” A slide overhead showcased the ground rules for the day, which included statements such as “dare to listen, dare to share, and dare to disagree.”

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Word cloud generated from live polling results at CRISPRcon Midwest.

CRISPRcon aimed to included voices beyond those represented by keynote speakers and panelists, so they incorporated live polling through an online app to keep the audience engaged and an active participant in the conversations throughout the day. From the opening remarks, it was clear that this conference would not just deliver on its promise of thoughtful conversation about the science, but build further understanding about the societal impacts of a rapidly advancing technology.

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All You Need is a Tether: Improving Repair Efficiency for CRISPR-Cas9 Gene Editing

Ribonucleoprotein complex with Cas9, guide RNA and donor ssDNA. Copyright Promega Corporation.

With the advent of genome editing using CRISPR-Cas9, researchers have been excited by the possibilities of precisely placed edits in cellular DNA. Any double-stranded break in DNA, like that induced by CRISPR-Cas9, is repaired by one of two pathways: Non-homologous end joining (NHEJ) or homology-directed repair (HDR). Using the NHEJ pathway results in short insertions or deletions (indels) at the break site, so the HDR pathway is preferred. However, the low efficiency of HDR recombination to insert exogenous sequences into the genome hampers its use. There have been many attempts at boosting HDR frequency, but the methods compromise cell growth and behave differently when used with various cell types and gene targets. The strategy employed by the authors of an article in Communications Biology tethered the DNA donor template to Cas9 complexed with the ribonucleoprotein and guide RNA, increasing the local concentration of the donor template at the break site and enhancing homology-directed repair. Continue reading “All You Need is a Tether: Improving Repair Efficiency for CRISPR-Cas9 Gene Editing”

NLRP3: The New Hope for Treating Chronic Inflammatory Diseases

Our innate immune system was meant to do good. Up until a century ago, most humans died from infectious diseases like diarrhea, tuberculosis and meningitis. Over millions of years, our immune system has evolved to fight these life-threatening infections from pathogens. As a result, we have developed a highly efficient response to these tiny invaders. But it seems that our immune system may be turning against us.

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Striking Fear into the Heart of Cardiovascular Disease Using Zebrafish and NanoLuc® Luciferase

Representative images of ApoB-LP localization in zebrafish across developmental, genetic, pharmacological and dietary manipulations.
Credit: Figure 5.D of The LipoGlo reporter system for sensitive and specific monitoring of atherogenic lipoproteins by James Thierer, Stephen C. Ekker and Steven A. Farber.
Article licensed under Creative Commons Attribution 4.0 International License.

Cardiovascular diseases, or CVDs, are collectively the most notorious gang of cold-blooded killers threatening human lives today. These unforgiving villains, including the likes of coronary heart disease, cerebrovascular disease and pulmonary embolisms, are jointly responsible for more deaths per year than any other source, securing their seat as the number one cause of human mortality on a global scale.

One of the trademarks of most CVDs is the thickening and stiffening of the arteries, a condition known as atherosclerosis. Atherosclerosis is characterized by the accumulation of cholesterol, fats and other substances, which together form plaques in and on the artery walls. These plaques clog or narrow your arteries until they completely block the flow of blood, and can no longer supply sufficient blood to your tissues and organs. Or the plaques can burst, setting off a disastrous chain reaction that begins with a blood clot, and often ends with a heart attack or stroke.

Given the global prevalence and magnitude of this problem, there is a significant and urgent demand for better ways to treat CVDs. In a recent study published in Nature Communications, researchers at the Carnegie Institution for Science, Johns Hopkins University and Mayo Clinic are taking the fight to CVDs through the study of low-density lipoproteins (LDLs), the particles responsible for shuttling bad cholesterol throughout the bloodstream.

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I Have My Luciferase Vector, Now What?

Choosing and Optimizing Transfection Methods

Here in Technical Services we often talk with researchers at the beginning of their project about how to carefully design and get started with their experiments. It is exciting when you have selected the Luciferase Reporter Vector(s) that will best suit your needs; you are going to make luminescent cells! But, how do you pick the best way to get the vector into your cells to express the reporter? What transfection reagent/method will work best for your cell type and experiment? Do you want to do transient (short-term) transfections, or are you going to establish a stable cell line?

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Prions Go Slow with ASOs: Experimental Treatment for ALS, Alzheimer’s and Other Prion-like Diseases

In the late-80’s through the 90’s, food and health agencies focused on a mysterious fatal brain disease that infected thousands of cattle. Bovine spongiform encephalitis—or “mad cow disease”—is caused by an infectious protein called a prion. Despite fears that tainted meat would cause the disease to spread to humans, mad cow disease never really made an impact on human health. However, forms of the prion disease such as Creutzfeldt-Jakob disease do affect humans.

In addition to Creutzfeldt-Jakob disease, many neurodegenerative diseases such as Alzheimer’s, Parkinson’s, Huntington’s and amyotrophic lateral sclerosis (ALS or Lou Gehrig’s disease) are now thought to be a result of prion-like activity. There is no cure for these diseases, however, new experimental treatment strategies might help slow the progression of neural degeneration.

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The tell-tale “holes” of prion infection in brain tissue.
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Immunotherapy—Don’t Forget the Microbiome

Bacteria make you sick. The idea that bacteria cause illness has become ingrained in modern society, made evident by every sign requiring employees to wash their hands before leaving a restroom and the frequent food recalls resulting from pathogens like E. coli. But a parallel idea has also taken hold. As microbiome research continues to reveal the important role that bacteria play in human health, we’re starting to see the ways that the microbiota of the human body may be as important as our genes or environment.

The story of how our microbiome affects our health continues to get more complex. For example, researchers are now beginning to understand that the composition of bacteria residing in your body can significantly impact the effects of therapeutic drugs. This is a new factor for optimizing drug response, compared to other considerations such as diet, interaction with other drugs, administration time and comorbidity, which have been understood much longer.

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