Daily Archives: April 3, 2015

Tuskegee Genome Workshop #39; 15 - What do they say?
Tuskegee University Plant Genome Biotechnology Workshop 2015 - High school teachers and students who participated in the summer event talk about their experience.

By: Channapatna Prakash

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Tuskegee Genome Workshop' 15 - What do they say? - Video

The gene editing method called CRISPR is already used in the lab to insert and remove genome defects in animal embryos

Genome editing has generated controversy, with unconfirmed reports of its use in human embryos. Credit: NIAID/Flickr

A tweak to a technique that edits DNA with pinpoint precision has boosted its ability to correct defective genes in people. Called CRISPR, the method is already used in the lab to insert and remove genome defects in animal embryos. But the genetic instructions for the machinery on which CRISPR reliesa gene-editing enzyme called Cas9 and RNA molecules that guide it to its targetare simply too large to be efficiently ferried into most of the human bodys cells.

This week, researchers report a possible way around that obstacle: a Cas9 enzyme that is encoded by a gene about three-quarters the size of the one currently used. The finding, published on 1April inNature, could open the door to new treatments for a host of genetic maladies (F. A. Ranetal. Naturehttp://dx.doi.org/10.1038/nature14299; 2015).

There are thousands of diseases in humans associated with specific genetic changes, says David Liu, a chemical biologist at Harvard University in Cambridge, Massachusetts, who was not involved in the latest study. A fairly large fraction of those have the potential to be addressed using genome editing.

Genome editing has generated controversy, with unconfirmed reports of its use in human embryos. Some scientists have expressed concern that the technique might be used by fertility doctors to edit the genes of human embryos before its safety is established (see alsoE.Lanphieret al. Nature519,410411; 2015). That concern is exacerbated by the fact that changes made by the procedure in embryos would be passed to all subsequent generations without giving anyone affected the opportunity to consent (seeNature519,272; 2015). But in the non-reproductive cells of children and adults, where intergenerational issues are not a concern, researchers and companies are already racing to develop CRISPR as a clinical tool.

The ethics of that pursuit may be more straightforward, but its execution can be harder than using CRISPR in embryos. An embryo consists of a small number of cells that give rise to a human. To edit the genome at that stage is simply a matter of injecting the necessary CRISPR components into a few cells. An adult human, however, is a mix of trillions of cells assembled into many different tissues. Researchers fret over how to target the CRISPR machinery to the specific cells where defective genes are disrupting physiological processes.

You can have the most optimal gene-editing system in the world, but if you cant deliver it to the proper cell type, its irrelevant, says Nessan Bermingham, chief executive of Intellia Therapeutics in Cambridge, Massachusetts, which aims to bring genome editing to the clinic. Were spending a tremendous amount of time working on it.

Snug fit Gene-therapy researchers often harness a virus called AAV to shuttle foreign genes into mature human cells. However, most laboratories use a gene encoding the Cas9 protein that is too large to fit in the snug confines of the AAV genome alongside the extra sequences necessary for Cas9 function.

Feng Zhang of the Broad Institute of MIT and Harvard in Cambridge, Massachusetts, and his colleagues decided to raid bacterial genomes for a solution, because the CRISPR system is derived from a process that bacteria use to snip unwanted DNA sequences out of their genomes. Zhangs team analysed genes encoding more than 600 Cas9 enzymes from hundreds of bacteria in search of a smaller version that could be packaged in AAV and delivered to mature cells.

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New Discovery Moves Gene Editing Closer to Use in Humans

Scientists in the BIDMC Cancer Research Institute discover that the non-coding BRAF pseudogene leads to the development of an aggressive lymphoma-like cancer in animal models

BOSTON -- Pseudogenes, a sub-class of long non-coding RNA (lncRNA) that developed from the genome's 20,000 protein-coding genes but lost the ability to produce proteins, have long been considered nothing more than genomic "junk." Yet the retention of these 20,000 mysterious remnants during evolution has suggested that they may in fact possess biological functions and contribute to the development of disease.

Now, a team led by investigators in the Cancer Research Institute at Beth Israel Deaconess Medical Center (BIDMC) has provided some of the first evidence that one of these non-coding "evolutionary relics" actually has a role in causing cancer.

In a new study in the journal Cell, publishing online today, the scientists report that independent of any other mutations, abnormal amounts of the BRAF pseudogene led to the development of an aggressive lymphoma-like disease in a mouse model, a discovery that suggests that pseudogenes may play a primary role in a variety of diseases. Importantly, the new discovery also suggests that with the addition of this vast "dark matter" the functional genome could be tremendously larger than previously thought - triple or quadruple its current known size.

"Our mouse model of the BRAF pseudogene developed cancer as rapidly and aggressively as it would if you were to express the protein-coding BRAF oncogene," explains senior author Pier Paolo Pandolfi, MD, PhD, Director of the Cancer Center and co-founder of the Institute for RNA Medicine (iRM) at BIDMC and George C. Reisman Professor of Medicine at Harvard Medical School. "It's remarkable that this very aggressive phenotype, resembling human diffuse large B-cell lymphoma, was driven by a piece of so-called 'junk RNA.' As attention turns to precision medicine and the tremendous promise of targeted cancer therapies, all of this vast non-coding material needs to be taken into account. In the past, we have found non-coding RNA to be overexpressed, or misexpressed, but because no one knew what to do with this information it was swept under the carpet. Now we can see that it plays a vital role. We have to study this material, we have to sequence it and we have to take advantage of the tremendous opportunity that it offers for cancer therapy."

The new discovery hinges on the concept of competing endogenous RNAs (ceRNA), a functional capability for pseudogenes first described by Pandolfi almost five years ago when his laboratory discovered that pseudogenes and other noncoding RNAs could act as "decoys" to divert and sequester tiny pieces of RNA known as microRNAs away from their protein-coding counterparts to regulate gene expression.

"Our discovery of these 'decoys' revealed a novel new role for messenger RNA, demonstrating that beyond serving as a genetic intermediary in the protein-making process, messenger RNAs could actually regulate expression of one another through this sophisticated new ceRNA 'language,'" says Pandolfi. The team demonstrated in cell culture experiments that when microRNAs were hindered in fulfilling their regulatory function by these microRNA decoys there could be severe consequences, including making cancer cells more aggressive.

In this new paper, the authors wanted to determine if this same ceRNA "cross talk" took place in a living organism -- and if it would result in similar consequences.

"We conducted a proof-of-principle experiment using the BRAF pseudogene," explains first author Florian Karreth, PhD, who conducted this work as a postdoctoral fellow in the Pandolfi laboratory. "We investigated whether this pseudogene exerts critical functions in the context of a whole organism and whether its disruption contributes to the development of disease." The investigators focused on the BRAF pseudogene because of its potential ability to regulate the levels of the BRAF protein, a well-known proto-oncogene linked to numerous types of cancer. In addition, says Karreth, the BRAF pseudogene is known to exist in both humans and mice.

The investigators began by testing the BRAF pseudogene in tissue culture. Their findings demonstrated that when overexpressed, the pseudogene did indeed operate as a microRNA decoy that increased the amounts of the BRAF protein. This, in turn, stimulated the MAP-kinase signaling cascade, a pathway through which the BRAF protein controls cell proliferation, differentiation and survival and which is commonly found to be hyperactive in cancer.

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An 'evolutionary relic' of the genome causes cancer