alanine – BioQuakes

Using CRISPR to target neurons

On March 12, 2019

A rat brain stained with protein and DNA.

Researchers from the University of Alabama at Birmingham have successfully used CRISPR to target neurons. With their novel approach, the team led by Jeremy Day was able to manipulate the function of neurons in vivo.

CRISPR, a self-defense system for bacteria against viral invaders, has become a very popular gene editing tool, as it allows researchers to make very targeted changes to an organism’s DNA. Normally using CRISPR-Cas9, the process involves a piece of guide RNA guiding Cas9 to the desired gene where it cuts it, rendering the gene inexpressible.

However, Day’s team used a different CRISPR mechanism, CRISPRa, which increases the expression of the desired gene. For their CRISPRa, they used CRISPR-dCas9, a CRISPR system with a deactivated Cas9, to which they attached transcriptional effectors. This allowed the guide RNA to guide the transcriptional effectors to a particular gene so it could be up-regulated, increasing its expression. In focusing on neurons, Day’s team targeted the promoter sequence for SYN genes, a common group of genes in the brain that code for proteins that regulate neurotransmitters, and designed their guide RNA accordingly.

After injecting their effector-coupled dCas9 system into live rats using viral hosts, the desired genes were successfully up-regulated, with the researchers viewing their new protein products after the fact through fluorescent markers in cell samples. Following this achievement, Day and his team expanded their CRISPR-dCas9 system, incorporating multiple guide RNAs into a single system to target multiple sections of DNA at the same time and using it analyze the complex Bdnf gene that has multiple promoters and plays a core role in brain function and development.

This innovative approach to targeting genes in the brain has far-reaching applications, allowing for versatile gene editing in live animals, which, in the words of Vanderbilt Brain Institute researcher Erin Calipari, “is going to give us an unprecedented view of the role of gene expression in behavior”.

From psychology to physiology and beyond, there is no doubt that this discovery’s molecular insight will give us a far greater understanding of the brain.

Programming protein pairs

By alanine

On January 14, 2019

In Student Post

Researchers from the University of Washington’s Institute of Protein Design have created a new method to engineer protein dimers, or pairs. Working alongside molecular biologists at Ohio State, the researchers have made it possible “to design proteins so they come together exactly how you want them to,” as the paper’s lead author explains.

Two proteins held together by DNA.

Before, researchers relied on DNA to engineer dimeric proteins, utilizing complementary strands to create helical proteins held together by the hydrogen bonds between base pairs. However, DNA-created proteins lack the functionality of highly active proteins like protease, while also being prone to interference during synthesis. So, longing to create these more complex protein assemblies, the researchers engineered a new way to make them.

Using a computer program called Rosetta, the researchers designed hydrogen bond networks for their desired protein complexes, creating complementary bond networks for each pair of amino acids. For this, Rosetta algorithmically determined the ideal shape of each amino acid chain, calculating the best way to balance out intermolecular forces and finding the resulting lowest energy level, the most probable state for each chain. Thus, the researchers could accurately design complementary protein structures, so the two parts would fit together exactly.

As a result, the researchers were able to create highly specific, more active protein dimers that form double helices unencumbered by DNA and do not form unwanted shapes or interfere with other proteins during synthesis.

This new method has the potential to “transform biomedical technology”, as scientists can now have much more control over protein interactions, potentially engineering bacteria to produce energy or designing protein machines to diagnose diseases, among many other tasks. As the researchers set their sights on more complicated, dynamic protein complexes, there is no telling what exciting discoveries await.

Genetically engineering the microbiome

By alanine

On November 19, 2018

In Student Post

Researchers from Harvard University have successfully taken the first steps in creating a synthetic microbiome. Using signaling between Salmonella Typhimurium and E. coli, the team was able to promote a new “genetic signal-transmission system” in mice.

A cluster of E. coli, a common species of gut bacteria.

With the hope of inducing interspecies bacterial communication, the researchers manipulated the bacterial signaling method of quorum sensing where bacteria receive and send signaling molecules in order to gauge their population density, performing a group behavior after reaching a certain threshold. By using the variant acyl-HSL quorum sensing, a version absent in mammals, the researchers were able to assess the feasibility of using a signaling system nonnative to its host.

In order to see if the two bacterial species successfully communicated, the researchers introduced both a signaler circuit and a responder circuit into the mice. The signaler circuit, put into Salmonella Typhimurium, contained a gene called luxI that, when turned on by a molecule called ATC, produced a quorum signaling molecule. This molecule was received by the bacteria with the responder circuit, E. coli, triggering a cro gene. This gene then turned on a LacZ gene, which caused the bacteria to turn blue when plated with special agar, and another cro gene, creating a loop that continuously activated the LacZ gene. This served as an indicator, as a blue glow would illustrate if the interspecies communication and the E.coli’s “memory” of it were successful.

After the mice were given the two edited strains of bacteria and placed in a container with ATC-infused water for two days, the researchers analyzed their fecal samples. They found that all of them turned blue, indicating that the genetically engineered signaling system was successful: the E. coli received and remembered a signal from Salmonella Typhimurium in response to an environmental factor. This effective engineered communication, as the Director of Harvard’s Wyss Institute for Biologically Inspired Engineering puts it, is a major step forward in “engineer[ing] intestinal microbes for the better while appreciating that they function as part of a complex community”.

With the basic principles of a synthetic microbiome a success, the researchers now want to experiment with new bacterial species and signaling molecules, bringing them closer towards their ultimate goal of engineering a gut microbiome that can perform tasks ranging from improving digestion to curing diseases. As the “next frontier in medicine [and] wellness,” the microbiome will no doubt be a key pillar of medicinal research for decades to come.

NIH program to create 3D map of human tissues

By alanine

On October 10, 2018

In Student Post

Nervous Tissue

A recent project from the National Institute of Health aims to build a 3D map of human tissues on a cellular level. Known as HuBMAP (Human BioMolecular Atlas Program), the project is largely seen as a successor to the Human Genome Project and will likely yield similarly incredible results.

With the goal of better understanding how cells organize and cooperate in tissues, the researchers involved in the project hope to be able to view the body with molecular level precision, understanding which “genes and proteins are activated in each part of the body” and the effects that has. Although it won’t map the entire body, the project will nevertheless present many challenges in dealing with big data, a very current issue in science research, as the researchers seek to map trillions of cells, compose high-resolution maps of them, and categorize those maps.

Caltech, one of the few institutions chosen for the program, is working on mapping the circulatory system, analyzing differences in the tissues of arteries, veins, and other components on a microscopic scale. Using a new imaging technique known as seqFISH, researchers at CalTech aim to analyze mRNA and in doing so pinpoint thousands of biomarkers across tissues in 3D.

In creating such a detailed and complex atlas of tissue maps, researchers hope to answer big questions in pathology and aging with one particular goal being to better understand how healthy tissues vary on a cellular level. As the project continues into the mid-2020s, one can only hope that HuBMAP enables us to fill critical gaps in our knowledge of cells, tissues, and health.

BioQuakes

AP Biology class blog for discussing current research in Biology

Author: alanine

Using CRISPR to target neurons

Programming protein pairs

Genetically engineering the microbiome

NIH program to create 3D map of human tissues

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