Category Archives: Genome

Octopus, squid, and cuttlefish are famous for engaging in complex behavior, from unlocking an aquarium tank and escaping to instantaneous skin camouflage to hide from predators. A new study suggests their evolutionary path to neural sophistication includes a novel mechanism: Prolific RNA editing at the expense of evolution in their genomic DNA.

The study, led by Joshua J.C. Rosenthal of the Marine Biological Laboratory (MBL), Woods Hole and Eli Eisenberg and Noa Liscovitch-Brauer of Tel Aviv University, is published this week in Cell.

The research builds on the scientists' prior discovery that squid display an extraordinarily high rate of editing in coding regions of their RNA - particularly in nervous system cells - which has the effect of diversifying the proteins that the cells can produce. (More than 60 percent of RNA transcripts in the squid brain are recoded by editing, while in humans or fruit flies, only a fraction of 1 percent of their RNAs have a recoding event.)

In the present study, the scientists found similarly high levels of RNA editing in three other "smart" cephalopod species (two octopus and one cuttlefish) and identified tens of thousands of evolutionarily conserved RNA recoding sites in this class of cephalopods, called coleoid. Editing is especially enriched in the coleoid nervous system, they found, affecting proteins that are the key players in neural excitability and neuronal morphology.

In contrast, RNA editing in the more primitive cephalopod Nautilus and in the mollusk Aplysia occurs at orders of magnitude lower levels than in the coleoids, they found. "This shows that high levels of RNA editing is not generally a molluscan thing; it's an invention of the coleoid cephalopods," Rosenthal says.

In mammals, very few RNA editing sites are conserved; they are not thought to be under natural selection. "There is something fundamentally different going on in these cephalopods where many of the editing events are highly conserved and show clear signs of selection," Rosenthal says.

The scientists also discovered a striking trade-off between high levels of RNA recoding and genomic evolution in these cephalopods. The most common form of RNA editing is carried out by ADAR enzymes, which require large structures (dsRNA) flanking the editing sites.

These structures, which can span hundreds of nucleotides, are conserved in the coleoid genome along with the editing sites themselves. The genetic mutation rate in these flanking regions is severely depressed, the team reported.

"The conclusion here is that in order to maintain this flexibility to edit RNA, the coleoids have had to give up the ability to evolve in the surrounding regions - a lot," Rosenthal says. "Mutation is usually thought of as the currency of natural selection, and these animals are suppressing that to maintain recoding flexibility at the RNA level."

Rosenthal and colleagues at the MBL are currently developing genetically tractable cephalopod model systems to explore the mechanisms and functional consequences of their prolific RNA editing.

"When do they turn it on, and under what environmental influences? It could be something as simple as temperature changes or as complicated as experience, a form of memory," he says.

Liscovitch-Brauer et al (2017) Trade-off between transcriptome plasticity and genome evolution in cephalopods. Cell DOI: 10.1016/j.cell.2017.03.025

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'Smart' cephalopods trade off genome evolution for prolific RNA editing - Space Daily

April 11, 2017

In recent years, science and the media have been buzzing with the term CRISPR. From speculation around reviving the woolly mammoth to promises of distant cures for cancer, the unproven potential for this genome editing tool has been stretched far and wide.

It's therefore no surprise that CRISPR has piqued the interest of many scientists and the public alike. So in this post we'll be exploring this innovation by answering some of the most common questions that pop up.

1. What is CRISPR?

CRISPR, or more precisely CRISPR-Cas9, is a molecular toolkit that scientists have developed to make precise edits to DNA our code of life. It was actually borrowed from bacteria, where in its original form it was used to protect microbes from attack by viruses.

2. How does it work?

The system is made up of 2 parts. The first is a strand of RNA DNA's chemical cousin which matches up with a region of DNA inside a cell that a scientist may want to target. This then acts as a shepherd to guide the second component a pair of 'molecular scissors' called Cas9 to the site of action, where it makes a snip across the DNA.

3. What can it do?

Once scientists have chopped their target region of DNA, a number of possibilities are opened up: they could disrupt the function of a particular gene, cut it out, make precise spelling changes to the DNA sequence, or slip in an entirely new gene. It's an extremely precise method of genetic modification.

4. Is it worth the hype?

CRISPR allows scientists to edit DNA in a way that's quicker, cheaper and more accurate than ever before. So it's an exciting development that's opening up new possibilities for scientists across the globe working in a number of different fields. But looking beyond the lab it's still very early days. Ideas for how the technology might be adapted to treat diseases are only just beginning to be considered. So it's important to be wary of premature promises made in the media when there is a lot of research to be done, and risks to be measured.

5. What are the concerns over safety as the tech develops?

Although CRISPR is hailed for its precision, concerns lie with what might happen if it misses its target, which it can. DNA is complex and many genes are intricately linked, so it could well be that modifying one gene has the scientists' desired outcome, but also inadvertently affects the function of other genes and molecules.

DNA is also written using an alphabet of just 4 chemical letters, meaning stretches of DNA that look very similar might both be targeted by CRISPR, which again may cause unintended effects. So scientists need to thoroughly scrutinise the consequences of their edits in these early lab development stages to ensure that they're not accidentally disrupting something important, which might not immediately be apparent.

6. How is it used in cancer research?

Cancer is caused by faulty genes, so recreating these in the lab with CRISPR allows researchers to explore the underlying biology of the disease and understand more about how it develops. That's what our scientists are doing for a type of brain tumour called glioblastoma.

Tweaking genes in cancer cells could also help identify those that are essential for the cells' survival, and therefore could be targeted with new treatments. On top of that, scientists could use the technique to explore ways that cancer cells become resistant to drugs, potentially opening up new ways to stop this from happening.

7. Could it help cure cancers?

Cancer isn't a single disease in fact, it's a group of more than 200 unique diseases so it's unlikely that any single treatment could act as a one-size-fits-all panacea. That includes CRISPR. And while there's no evidence yet that CRISPR can be used to treat cancer, it's possible that as the technology develops it could be used in treatments in some way. The most promising idea so far is to use it in cell therapy, where patients' own immune cells would be taken out and tweaked, giving them a 'power-up' so that they can better attack the cancer when given back to the patient. But this idea still needs testing in clinical trials.

8. What can't it do (yet)?

With the advent of gene editing came the idea that this technique could potentially be used to correct faulty, disease-causing genes in people, therefore curing their illness. This remains a long way off, and would be an incredibly complex area to study, but it's not impossible. Much more research is needed first and where the desired edits might involve correcting inherited faulty genes, there are huge ethical questions to address, particularly around editing human embryos. These kinds of public debates, discussions and expert recommendations are already underway.

9. So, where are we now?

In the context of cancer, CRISPR is beginning to move from lab bench to bedside. Last year scientists in China began trialling CRISPR-edited immune cells in lung cancer patients, where they'd snipped out a gene that produces a stop signal, called PD-1, for the immune system. They plan to test if this edit will boost the cells' cancer-killing abilities, but we won't know the results for a while.

A team in the US is also nipping at their heels, launching a similar trial this year but for several different cancers.

These could mark the beginnings of a new wave of cancer treatment. And those clinical trials will hopefully provide some early answers.

Whether or not CRISPR will ultimately match its promise is unknown. But it's an exciting time for science.

Of that there is no doubt.

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Researchers at the University of Alberta have demystified the way that polar bears search for their typical prey of ringed seals. The answer, it turns out, is simple: they follow their nose using the power of wind.

Asian elephants are able to recognise their bodies as obstacles to success in problem-solving, further strengthening evidence of their intelligence and self-awareness, according to a new study from the University of Cambridge.

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Nine burning questions about CRISPR genome editing answered - Phys.Org

Mapping a prostate tumour for the first time was a long, slow and difficult process

In a world first, Australian researchers have mapped the entire genome of a prostate tumour, providing a new lens through which to view this disease.

They mapped the most commonly diagnosed grade of prostate cancer. It was a tumour that scored 7 on the standard Gleason score and is clinically known to be highly unpredictable.

The mapping process, which useda biopsy sample taken from a patient, was long and slow.

It required the researchers to get DNA out of tissue without destroying it. Previously this had not been achieved in humans.

The results of their study of this single tumour are published in Oncotarget,a journal aimed at doctors and scientists.

Prostate cancer is the most commonly diagnosed cancer in Australian men and this work is proof of principle that next-generation mapping can provide insights into its subtypes.

The information gained by such mapping could be used to characterise an individual's tumour and reveal previously unrecognised information so treatment can be more targeted.

Conducted at Sydney's Garvan Institute of Medical Research, the mapping revealed previously undetected levels of DNA changes linked to the disease.

It uncovered 10 times more large-scale DNA rearrangements than have previously been detected in prostate cancer and identified 15 new potential drivers of this cancer.

"Although we've been researching prostate cancer for many years, very little is understood about what drives these tumours," says study leader, Professor Vanessa Hayes, Head of Garvan's Human Comparative and Prostate Cancer Genomics Laboratory.

"One of the biggest clinical challenges is distinguishing which cancers are going to spread and become life-threatening, and which patients could be spared harsh treatment they might not need.

"To have any hope of targeting treatment in this way, we first need to understand the genetic drivers of each individual tumour. "

The researchers used new mapping technology in tandem with whole genome sequencing to uncover the most complete picture to date of the prostate cancer genomic landscape.

While genomic sequencing is a close up exercise which reads each letter of a genetic code, mapping takes a few steps back and provides a bigger picture. It gives a bird's eye view, orientating the sequence in its context.

She says prostate cancer has unique features.

"From previous genome sequencing studies we know it has very few small genetic changes, but rather, is more likely driven by large complex rearrangements of DNA within the genome. "

"This is different to most cancers, which are driven by small DNA mutations in a number of key genes."

"Until now, we had no way of observing these DNA rearrangements or structural variants in prostate cancer."

Professor Hayes says the synergy with whole genome sequencing was very important.

"We could not have done this with sequencing technology alone. Whole genome sequencing is invaluable in identifying small DNA mutations, but it may not detect when a gene has been completely deleted, transferred to another chromosome, or multiplied many times - which is what we see here."

"Using next-generation mapping, we saw huge amounts of large-scale rearrangements, and genome sequencing then enabled us to identify the genes affected by these rearrangements."

"Several cancer-promoting genes were multiplied many times, increasing their potency, and potentially driving this prostate tumour."

"Whole genome sequencing opened a huge number of doors for our understanding of prostate cancer next-generation mapping just doubled the number of doors," says Prof Hayes.

Her team was first in Australia to obtain next-generation mapping technology, and first in the world to apply it to understanding an individual tumour.

"I believe that in the future this technology will complement next generation sequencing as a key to personalised medicine for prostate cancer."

The study was performed as part of the Prostate Cancer Metastasis (ProMis) program, an Australian-led international initiative. Since it began, mapping technology has improved and is now faster.

The Garvan team has since mapped a further four tumours which will be the subject of another paper. Professor Hayes says this new work confirms the significance of these large DNA changes detected in the first study.

When it comes to mapping technology for cancer in humans, Australia is a world leader.

"This is a very promising research breakthrough," says Professor Allan Spigelman, Director of Cancer Genetics at Sydney's St Vincents' Hospital.

"In time, it will complement some current treatment options that are based on genetic testing of blood samples. Using prostate cancer tissue genetic analysis may hopefully lead to even more precise and targeted treatments."

Professor Spigelman, who conducts cancer genetics services across NSW, say at present men with prostate cancer can have genetic testing to see if they have a good chance of responding to particular medication.

"Current cancer gene testing of blood samples target DNA repair genes such as BRCA2."

"Detection of a mutation here opens up novel drug treatments to which those carrying mutations in that gene respond best."

*Jill Margo is an adjunct associate professor at the University of NSW

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Australian researchers first to map entire prostate cancer genome - The Australian Financial Review

The Ewing Marion Kauffman Foundations vice president of research and policy is departing the organization for a San Francisco-based firm focused on startup ecosystem research.

Dane Stangler

A 12-year veteran at the Kansas City-based foundation, Dane Stangler is now the head of policy at Startup Genome, a company that researches ecosystems and advises policymakers to increase the success rate of startups and accelerate economic growth. Starting with Kauffman in 2004 as a senior analyst, Stangler worked up the ranks at the Kauffman Foundation and in 2014 was named vice president of research and policy. In addition to representing the foundation at conferences around the U.S., hes published in such publications as the Wall Street Journal and Huffington Post.

At Startup Genome, Stangler will work on the firms Lifecycle Model, which aims to help entrepreneurial ecosystems around the world.

I am absolutely thrilled to join Startup Genomes global platform and help contribute to their mission of increasing startup success around the world, bringing more people and places into the startup revolution, Stangler said in a release. Too many places continue to take public and private actions that harm or drive away startups. Startups deserve better. We want to make sure that policy everywhere is the most conducive it can be to startups.Startup Genomes mission is to empower cities around the world to capture their fair share of the new economy by accelerating the economic growth of startup ecosystems through benchmarking, networking, and exposure. The firm conducts research with more than 10,000 startups each year and aims to build consensus for action on key challenges.Kauffman Foundation CEO Wendy Guillies said shes excited about Stanglers future.

This is a natural fit for Dane, leveraging both his talent and dedication to helping entrepreneurs succeed, Guillies said in a release. We are proud of his accomplishments at the Foundation, and we look forward to seeing him bring his wealth of experience to help Startup Genome strengthen ecosystems around the world.

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Kauffman exec departs for leadership role at Startup Genome - Startland News

CAMBRIDGE, Mass.--(BUSINESS WIRE)--Seven Bridges, the biomedical data analysis company, today announced that it has made the Simons Genome Diversity Project (SGDP) dataset available for analysis by researchers via the Seven Bridges Platform.

The SGDP is the largest dataset of human genetic variation ever collected, including whole genomes from 300 individuals representing 142 diverse populations across the globe. The SGDP dataset is now available for Seven Bridges Platform users to analyze in conjunction with their own data and other large datasets including The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia (CCLE). The Platform is used by thousands of researchers around the world to drive research and development in the worlds largest biopharmaceutical organizations.

Partnering with Seven Bridges will put this diverse and unique dataset into the hands of more researchers, in turn, speeding the discovery process, said Dr. David Reich of Harvard Medical School, one of the directors of the project. One of the most important components of scientific practice is the ability of scientists to replicate analysis, reanalyze data, build on it and come to their own conclusions. The Seven Bridges Platform and tools provide a new way for researchers all over the world to leverage our data and make new discoveries.

The SGDP dataset is particularly valuable to researchers because it differs from most other large-scale genomic datasets. The Simons Foundation selected samples with the explicit intent of capturing as much geographic, anthropological, and linguistic diversity as possible. As a result, this dataset captures modern human genetic diversity that is not well represented in other genomics datasets. As a result, the SGDP dataset provides valuable guidance to understand evolutionary pressures towards identifying important parameters in the search for disease-related genes. Once researchers identify data of interest, it can be immediately imported into their project tobuild reproducible bioinformatic analyses.

The Simons Foundation has long been committed to advancing the frontiers of scientific research, with a focus on creating collaborations that will generate discovery for years to come. said Brandi Davis-Dusenbery, CEO of Seven Bridges. The release of SGDP on our Platform will help researchers around the world more effectively use this powerful dataset.

More information on the Simons Genome Diversity Project dataset is available on the Seven Bridges blog.

About Seven Bridges Seven Bridges is the biomedical data analysis company accelerating breakthroughs in genomics research for cancer, drug development and precision medicine. The scalable, cloud-based Seven Bridges Platform empowers rapid, collaborative analysis of millions of genomes in concert with other forms of biomedical data. Thousands of researchers in government, biotech, pharmaceutical and academic labs use Seven Bridges, including three of the largest genomics projects in the world: U.S. National Cancer Institutes Cancer Genomics Cloud pilot, the Million Veteran Program, and Genomics Englands 100,000 Genomes Project. As the NIHs only commercial Trusted Partner, Seven Bridges authenticates and authorizes access to one of the worlds largest cancer genomics dataset. Named one of the worlds smartest companies by MIT Technology Review, Seven Bridges has offices in Cambridge, Mass.; Belgrade; London; Istanbul; and San Francisco.

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Seven Bridges Brings The Simons Foundation's Genome Diversity Project Dataset to the Cloud - Business Wire (press release)

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Grand View Research, Inc. Market Research And Consulting.

According to the report published by Grand View Research Clustered Regularly Interspaced Short Palindromic Repeats CRISPR and CRISPR-associated (Cas) genes market is anticipated to reach USD 4.09 billion by 2025.Possibility of rewriting the host DNA through the virtue of Cas9 by introduction of major modifications can be attributed for rising adoption of technology.

Clustered Regularly Interspaced Short Palindromic RepeatsCRISPR and CRISPR-Associated (Cas) Genes Marketis anticipated to reach USD 4.09 billion by 2025, according to a new report by Grand View Research, Inc. This genome editing principle spans almost every industry that involves biological systems. The rising adoption of technology in different areas associated with biotechnology is anticipated to drive industrial growth of the technology substantially in the coming years.

Possibility of rewriting the host DNA through the virtue of Cas9 by introduction of major modifications can be attributed for rising adoption of technology. These modifications include inversion, deletions, knockouts, translocations, and gene replacement.

Moreover, application of the technology as a qualitative as well as quantitative tool in plant genome editing is expected to propel growth. The technique holds the potential for producing plants with mutations linked to other disciplines of science such as disease resistance, biofuel production, synthetic biology, phytoremediation and abiotic stress tolerance.

Combination of clustered regularly interspaced short palindromic repeats and sequencing technology enables high-throughput analysis of gene regulation thereby resulting to enhancement in genomics sector. The aforementioned combination is applicable in the epigenetic study of diseases such as leukaemia.

However, off-target effects associated with the implementation of CRISPR is anticipated to impede growth in the coming years. These effects include improper concentration ratio between Cas9 and single guide RNA that may result into off-target cleavage.

Full research report on CRISPR and CRISPR-Associated (Cas) Genes Market: http://www.grandviewresearch.com/industry-analysis/crispr-associated-cas-genes-market

Further Key Findings from the Report Suggest:

View more reports of this category by Grand View Research at: http://www.grandviewresearch.com/industry/biotechnology

Grand View Research has segmented the CRISPR and Cas Genes market on the basis of product, application, end-use, and region:

CRISPR and Cas Genes Product Outlook (Revenue, USD Million, 2014 2025)

CRISPR and Cas Genes Application Outlook (Revenue, USD Million, 2014 2025)

CRISPR and Cas Genes End-use Outlook (Revenue, USD Million, 2014 2025)

CRISPR and Cas Genes Regional Outlook (Revenue, USD Million, 2014 2025)

Access press release of this research report by Grand View Research: http://www.grandviewresearch.com/press-release/global-crispr-associated-cas-genes-market

About Grand View Research Grand View Research, Inc. is a U.S. based market research and consulting company, registered in the State of California and headquartered in San Francisco. Thecompany provides syndicated research reports, customized research reports, and consulting services. To help clients make informed business decisions, we offer market intelligence studies ensuring relevant and fact-based research across a range of industries, from technology to chemicals, materials and healthcare.

For more information:www.grandviewresearch.com

Media Contact Company Name: Grand View Research, Inc. Contact Person: Sherry James, Corporate Sales Specialist U.S.A. Email: sales@grandviewresearch.com Phone: 1-415-349-0058, Toll Free: 1-888-202-9519 Address:28 2nd Street, Suite 3036 City: San Francisco State: California Country: United States Website: http://www.grandviewresearch.com/industry-analysis/crispr-associated-cas-genes-market

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CRISPR And Cas Genes Market Is Anticipated To Drive Industrial ... - satPRnews (press release)

A new method lets researchers quicklyscreen the non-coding DNA of the human genome for links to diseases that are driven by changes in gene regulation.

The technique could revolutionize modern medicines understanding of the genetically inherited risks of developing heart disease, diabetes, cancer, neurological disorders, and others, and lead to new treatments.

Identifying single mutations that cause rare, devastating diseases like muscular dystrophy has become relatively straightforward, says Charles Gersbach, the associate professor of biomedical engineering at Duke University. But more common diseases that run in families often involve lots of genes as well as genetic reactions to environmental factors. Its a much more complicated story, and weve been wanting a way to better understand it. Now weve found a way.

As reported in Nature Biotechnology, the new technique relies on the gene-hacking system called CRISPR/Cas9. Originally discovered as a natural antiviral defense mechanism in bacteria, the system recognizes and homes in on the genetic code of previous intruders and then chops up their DNA. In the past several years, researchers have harnessed this biologic system to precisely cut and paste DNA sequences in living organisms.

In the current study, researchers added molecular machinery that can control gene activity by manipulating the web of biomolecules that determines which genes each cell activates and to what degree.

With the new tool, Gersbach and his colleagues are exploring the 98 percent of our genetic code often referred to as the dark matter of the genome.

Only a small fraction of our genome encodes instructions to make proteins that guide cellular activity, says Tyler Klann, the biomedical engineering graduate student who led the work in Gersbachs lab. But more than 90 percent of the genetic variation in the human population that is associated with common disease falls outside of those genes. We set out to develop a technology to map this part of the genome and understand what it is doing.

The answer, says Klann, lies with promoters and enhancers. Promoters sit directly next to the genes they control. Enhancers, however, which modulate promoters, can be just about anywhere due to the genomes complex 3D geometry, making it difficult to discern what theyre actually doing.

If an enhancer is dialing a promoter up or down by 10 or 20 percent, that could logically explain a small genetic contribution to cardiovascular disease, for example, says Gersbach. With this CRISPR-based system, we can more strongly turn these enhancers on and off to see exactly what effect theyre having on the cell. By developing therapies that more dramatically affect these targets in the right direction, we could have a significant effect on the corresponding disease.

Thats all well and good for exploring the regions of the genome that researchers have already identified as being linked to diseases, but there are potentially millions of sites in the genome with unknown functions. To dive down the dark genome rabbit hole, Gersbach turned to colleagues Greg Crawford, associate professor of pediatrics and medical genetics, and Tim Reddy, assistant professor of bioinformatics and biostatistics. All three professors work in the Duke Center for Genomic and Computational Biology.

Crawford developed a way of determining which sections of DNA are open for business. That is, which sections are not tightly packed away, providing access for interactions with biomachinery such as RNA and proteins. These sites, the researchers reason, are the most likely to be contributing to a cells activity in some way. Reddy has been developing computational tools for interpreting these large genomic data sets.

Over the past decade, Crawford has scanned hundreds of types of cells and tissues affected by various diseases and drugs and come up with a list of more than 2 million potentially important sites in the dark genomeclearly far too many to investigate one at a time.

In the new study, Crawford, Reddy, and Gersbach demonstrate a high-throughput screening method to investigate many of these potentially important genetic sequences in short order. While these initial studies screened hundreds of these sites across millions of base pairs of the genome, the researchers are now working to scale this up 100- to 1,000-fold.

Small molecules can target proteins and RNA interference targets RNA, but we needed something to go in and modulate the non-coding part of the genome, says Crawford. Up until now, we didnt have that.

The method starts by delivering millions of CRISPR systems loaded into viruses, each targeting a different genetic point of interest, to millions of cells in a single dish. After ensuring each cell receives only one virus, the team screens them for changes in their gene expression or cellular functions.

For example, someone researching diabetes could do this with pancreatic cells and watch for changes in insulin production. Those cells that show interesting alterations are then isolated and sequenced to determine which stretch of DNA the CRISPR affected, revealing a new genetic piece of the diabetes puzzle.

The technique is already producing results, identifying previously known genetic regulatory elements while also spotting a few new ones. The results also show it can be used to turn genes either on or off, which is superior to other tools for studying biology that only turn genes off. Different cell types also produced differentbut partially overlappingresults, highlighting the biological complexity in gene regulation and disease that can be interrogated with this technology.

Now that we have this tool, we can go in and annotate the functions of these previously unknown but important stretches of our genome, says Gersbach. With so many places to look, and the ability to do it quickly and robustly, well undoubtedly find new segments that are important for disease, which will provide new avenues for developing therapeutics.

The Thorek Memorial Foundation, the National Institutes of Health, and the National Science Foundation supported the work.

Source: Duke University

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How scientists explore our genome's 'dark matter' - Futurity: Research News

Researchers have developed a method to swiftly screen the non-coding DNA of the human genome for links to diseases that are driven by changes in gene regulation. The technique could revolutionize modern medicines understanding of the genetically inherited risks of developing heart disease, diabetes, cancer, neurological disorders and others, and lead to new treatments.

The study appeared online in Nature Biotechnology on April 3, 2017.

Identifying single mutations that cause rare, devastating diseases like muscular dystrophy has become relatively straightforward, said Charles Gersbach, the Rooney Family Associate Professor of Biomedical Engineering at Duke University. But more common diseases that run in families often involve lots of genes as well as genetic reactions to environmental factors. Its a much more complicated story, and weve been wanting a way to better understand it. Now weve found a way.

The new technique relies on the gene-hacking system called CRISPR/Cas9. Originally discovered as a natural antiviral defense mechanism in bacteria, the system recognizes and homes in on the genetic code of previous intruders and then chops up their DNA. In the past several years, researchers have harnessed this biologic system to precisely cut and paste DNA sequences in living organisms.

In the current study, researchers added molecular machinery that can control gene activity by manipulating the web of biomolecules that determines which genes each cell activates and to what degree.

With the new tool, Gersbach and his colleagues are exploring the 98 percent of our genetic code often referred to as the dark matter of the genome.

Only a small fraction of our genome encodes instructions to make proteins that guide cellular activity, said Tyler Klann, the biomedical engineering graduate student who led the work in Gersbachs lab. But more than 90 percent of the genetic variation in the human population that is associated with common disease falls outside of those genes. We set out to develop a technology to map this part of the genome and understand what it is doing.

The answer, says Klann, lies with promoters and enhancers. Promoters sit directly next to the genes they control. Enhancers, however, which modulate promoters, can be just about anywhere due to the genomes complex 3D geometry, making it difficult to discern what theyre actually doing.

If an enhancer is dialing a promoter up or down by 10 or 20 percent, that could logically explain a small genetic contribution to cardiovascular disease, for example, said Gersbach. With this CRISPR-based system, we can more strongly turn these enhancers on and off to see exactly what effect theyre having on the cell. By developing therapies that more dramatically affect these targets in the right direction, we could have a significant effect on the corresponding disease.

Thats all well and good for exploring the regions of the genome that researchers have already identified as being linked to diseases, but there are potentially millions of sites in the genome with unknown functions. To dive down the dark genome rabbit hole, Gersbach turned to colleagues Greg Crawford, associate professor of pediatrics and medical genetics, and Tim Reddy, assistant professor of bioinformatics and biostatistics. All three professors work together in the Duke Center for Genomic and Computational Biology.

Crawford developed a way of determining which sections of DNA are open for business. That is, which sections are not tightly packed away, providing access for interactions with biomachinery such as RNA and proteins. These sites, the researchers reason, are the most likely to be contributing to a cells activity in some way. Reddy has been developing computational tools for interpreting these large genomic data sets.

Over the past decade, Crawford has scanned hundreds of types of cells and tissues affected by various diseases and drugs and come up with a list of more than 2 million potentially important sites in the dark genomeclearly far too many to investigate one at a time. In the new study, Crawford, Reddy and Gersbach demonstrate a high-throughput screening method to investigate many of these potentially important genetic sequences in short order. While these initial studies screened hundreds of these sites across millions of base pairs of the genome, the researchers are now working to scale this up 100- to 1000-fold.

Small molecules can target proteins and RNA interference targets RNA, but we needed something to go in and modulate the non-coding part of the genome, said Crawford. Up until now, we didnt have that.

The method starts by delivering millions of CRISPR systems loaded into viruses, each targeting a different genetic point of interest, to millions of cells in a single dish. After ensuring each cell receives only one virus, the team screens them for changes in their gene expression or cellular functions.

For example, someone researching diabetes could do this with pancreatic cells and watch for changes in insulin production. Those cells that show interesting alterations are then isolated and sequenced to determine which stretch of DNA the CRISPR affected, revealing a new genetic piece of the diabetes puzzle.

The technique is already producing results, identifying previously known genetic regulatory elements while also spotting a few new ones. The results also showed it can be used to turn genes either on or off, which is superior to other tools for studying biology which only turn genes off. Different cell types also produced differentbut partially overlappingresults, highlighting the biological complexity in gene regulation and disease that can be interrogated with this technology.

Now that we have this tool, we can go in and annotate the functions of these previously unknown but important stretches of our genome, said Gersbach. With so many places to look, and the ability to do it quickly and robustly, well undoubtedly find new segments that are important for disease, which will provide new avenues for developing therapeutics.

This work was supported by the Thorek Memorial Foundation, the National Institutes of Health (R01DA036865, U01HG007900, DP2OD008586, P30AR066527, T32GM008555, R41GM119914) and the National Science Foundation (CBET-1151035).

CRISPRCas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Tyler S. Klann, Joshua B. Black, Malathi Chellappan, Alexias Safi, Lingyun Song, Isaac B. Hilton, Gregory E. Crawford, Timothy E. Reddy and Charles A. Gersbach. Nature Biotechnology, April 3, 2017. DOI: 10.1038/nbt.3853

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Screening the Human Genome's 'Dark Matter' - Duke Today

Schultz, et. al., Science

Viruses tend to have stripped-down genomes, carrying just enough genes to take over a cell and make lots more copies. Ebola, for example, carries a total of just seven genes, allowing new copies to be made with little fuss. There are a few exceptionsviruses like herpes with complex life cyclesbut even the biggest of the viruses we knew about had only a few hundred genes.

All that changed a bit more than a decade ago, when researchers discovered the Mimivirus, which had a genome bigger than some bacteria and carried many genes for functions that are normally provided by host proteins. The huge genomes and strange behavior of the viruses led their discoverers to propose that they weren't just odd offshoots that preyed upon liferather, they might have played a critical role in boosting life's complexity.

Now, researchers have discovered a new family of giant viruses, related to the Mimiviruses but distinct in a number of ways. And a careful analysis of their genes suggests they, and all other giant viruses, have been put together through relatively recent evolution. The work argues very strongly against these viruses playing a key role in life's diversification.

The Mimiviruses contain many of the genes needed to read DNA and use the information to make proteins; most other viruses rely entirely on the system that their host cells use. They also set up an odd "virus factory" inside cells they infect, which appears physically distinct from the rest of the cell's contents.

To the people that discovered the virus, this looked almost as if the virus were setting up its own nucleus, the place where cells normally store their DNA. So they suggested this might be how cells ended up with a nucleus in the first place: a virus set up shop in what had been a simple cell, and never left. Additional complexity evolved over time, but many of the virus' genes are still around in complex cells like our own.

While intriguing, the proposal rested on the idea that the giant viruses are a distinct lineage that has been around since the main branches of the tree of life first started. And, from a genetic standpoint, this seemed plausible; many of the genes the viruses carry were either previously unknown or not closely related to the genes of the host they preyed upon.

That idea was put to a test by the combination of an Austrian-US research collaboration and a sewage plant. Samples from the wastewater treatment plant at Klosterneuburg, Austria were subjected to what's called metagenome analysis. Rather than trying to culture everything that grew in the waste, the authors simply isolated DNA from it and started sequencing. Computers can then search for pieces that overlap, gradually building up individual genomes out of the random parts.

This turned up Klosneuvirus, another giant virus with a genome 1.6 million base pairs long. Electron microscopy of the sewage water then revealed giant viruses were present. Struck by this success, the authors then started searching through other metagenome data sets. This search put together three additional giant virus genomes, belonging to Catovirus, Hokovirus, and Indivirus. Combined, the new viruses add 2,500 additional gene families to the ones previously found in giant viruses.

An evolutionary comparison showed that these viruses were closely related to the Mimivirus family but formed a distinct branch. And compared to the Mimiviruses, they had an even larger collection of genes needed for proteinmanufacture, being able to incorporate 14 of the 20 different amino acids into proteins without any help from the host.

If giant viruses were involved in the origin of life, then the new sequences should shed some light on that. The hypothesis has some consequencesthe viruses should share a core set of genes that are distinct, forming its own domain on the tree of life.

The new study finds very little evidence of that. Instead, as noted above, the new viruses have a lot more protein-manufacturing genes than the Mimiviruses. When the authors analyzed each of these genes individually, they were typically most closely related to a species with a complex cell, rather than another virus. Most of these branches were fairly recent, as well.

In fact, of more than 20 instances of a specific type of gene in the Klosneuvirus, only seven were shared with all the other giant viruses. Only three of those appear to date back to the ancestor of all giant viruses. And only two appear to be distinct enough that they could belong to a distinct branch of the tree of life. The same pattern was apparent in all the other classes of genes involved in making proteins. And, critically, some key components that are used by all branches of life are missing (like the RNAs that are part of the ribosomes, which catalyze protein production.)

In fact, the Klosneuvirus family themselves look like they were stitched together from spare parts. Collectively, the four viruses share 355 genes with species with complex cells. But only 12 of those genes are found in all four of the viruses. Most of them instead seem to have been picked up after the individual virus species split off.

So, the authors propose what they call an "accordion model" of the viruses' evolution. Under some circumstances, the virus goes through periods where it loses genes, slimming down in size somewhat. In other times, the virus picks up new genes, with a preference for certain functions (like preparing amino acids for incorporation into proteins). At the moment, we know too little about the viruses to guess as to what pressures might drive either the expansion or the contraction.

Although the authors don't say as much, however, the fact that they're giant viruses probably makes a big difference in terms of whether that expansion/contraction can happen at all. Many smaller viruses make coats to contain their genetic material that have hard size limitsgeometry dictates that the proteins that form the coat can only come together in specific ways. This size limit, in turn, limits the amount of genetic material that can be squeezed inside. The giant viruses make a correspondingly giant coat, one that may have a lot more flexibility in terms of how much material it can hold.

Science, 2017. DOI: 10.1126/science.aal4657 (About DOIs).

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New giant viruses suggest their genomes expanded like an ... - Ars Technica

April 6, 2017 A depiction of the double helical structure of DNA. Its four coding units (A, T, C, G) are color-coded in pink, orange, purple and yellow. Credit: NHGRI

The DNA molecules in each one of the cells in a person's body, if laid end to end, would measure approximately two metres in length. Remarkably, however, cells are able to fold and compact their genetic material in the confined space of the nucleus, which spans only a few micrometres. Importantly, the compaction and arrangement of the genome inside the nucleus needs to be achieved in an ordered fashion that still allows cells to access the genetic information appropriately, for example to produce messenger RNAs for specific proteins, or to replicate the genetic material prior to cell division. When mutations occur that disrupt features associated with the spatial organisation of the genome, this leads to developmental disorders and cancer.

Scientists have had a long-standing interest in examining the spatial organisation of the genome in the cell's nucleus, mostly using microscopy techniques. Recent advances in genomic techniques to measure the 3D organisation of the genome have allowed for an increased resolution of this organisation. However, when the genome gains 3D organisation during development is not known. Now, using early development fruit fly embryos and genomic techniques to measure 3D genome organisation, scientists of the Research Group 'Regulatory Genomics' at the Max Planck Institute for Molecular Biomedicine in Muenster have shown that the 3D organisation of the genome emerges when the early embryo switches on its own genetic programme.

A common image of the cell's genetic material is the rod-like structures of mitotic chromosomes. However, those only exist while cells are undergoing cell division. The rest of the time, the genetic material is found in the form of chromatin fibres - DNA molecules densely wrapped around histone proteins - which are less densely compacted than mitotic chromosomes and occupy the nuclear space.

"One could think of this as a plate of spaghetti, where each individual piece of pasta would correspond to the DNA molecule in each chromosome", says Juanma Vaquerizas, head of the Max Planck Research Group 'Regulatory Genomics' at the Max Planck Institute for Molecular Biomedicine, who led the study. "A fundamental question in the field was whether each spaghetti would randomly mingle with other pieces of pasta or whether they would occupy a defined space within the plate."

Using microscopy approaches, scientists had determined before that the location of chromatin in the nucleus was not random, and recent advances in our ability to measure chromatin architecture have shown that finer structures, called topologically associating domains (TADs), form part of the basic functional units that determine the 3D organisation of the genome. However, a very puzzling observation has been that when the TAD organisation of the genome is examined in different cell types in an organism or in conserved regions of the DNA between species, this seems to be very similar across samples, despite different parts of the genome being actively used in different cell types. This prompted Clemens Hug and Juanma Vaquerizas to address the question of when during organismal development chromatin architecture is established.

The team turned to early development of fruit flies to perform their experiments. "An amazing feature about fruit fly embryonic development is that upon fertilization, the nuclei synchronously divide every 10-15 minutes for thirteen times without gene activation", says Vaquerizas. Maternally deposited mRNAs and proteins make sure that differentiation and development occur during those initial nuclear cycles. Then, at nuclear cycle 14 - only 2,5 hours after fertilization - the embryonic genome is activated. "Thus, in fruit flies, we can accurately study early chromatin organization at a high temporal resolution", says Vaquerizas.

The choice of organism and its developmental timing proved critical for the researchers' experiments, since this allowed them to examine 3D genome organisation in nuclei at a stage when transcription is naturally not occurring, and by doing so, decouple genome organisation from the effects of transcription.

By using state-of the-art genomic analyses, the scientists were able to study chromatin organization at a very high spatial resolution. Clemens Hug, PhD student and first author of the study, explains the method they used: "The so-called in situ Hi-C technique allows us to accurately identify those parts of the DNA that interact with each other in the three-dimensional nuclear space and the extent of interaction throughout the genome. We are therefore able to capture the 3D organization of the chromatin at a certain time point and can reveal changes in organisation across early development stages." Strikingly, the team found that at early stages of development the genome lacks defined higher-order chromatin organisation, and that 3D architecture progressively emerges in later stages.

"We found that TAD boundaries - defining functionally distinct chromatin units - arise when the first zygotic genes are transcribed. The number of TAD boundaries reaches a plateau when the complete zygotic genome has been activated", says Hug. "These boundaries are occupied by housekeeping genes that are constantly transcribed in all cell types. Once established, these are maintained throughout development." This is an important finding since it helps explain why the TAD organisation of genomes is similar across tissue types and evolutionary conserved regions between species.

However, the scientists could demonstrate that the establishment of TAD boundaries is independent of transcription itself, despite being associated with transcriptionally active regions. "This is of interest since it suggests that the machinery or mechanisms leading to transcription might play a role in TAD boundary establishment", says Hug. The scientists observed that Zelda, a pioneer transcription factor protein that opens the chromatin so that the transcription machinery can access the DNA, is necessary to establish some TAD boundaries. "We therefore think that Zelda and maybe other proteins with a similar function, in concert with RNA Pol II, create the TAD boundaries and thus are responsible for the 3D chromatin architecture", says Hug.

"When the proteins that determine TAD boundaries - and thus are critical for the chromatin architecture - are disrupted, this can result in distinct developmental disorders and cancer", says Vaquerizas. "Our newly gained insights into how the 3D chromatin architecture is established and maintained will thus have a major impact on further studies looking at its impact on gene expression during development and disease."

Explore further: Scientists reveal hidden structures in bacterial DNA

More information: Clemens B. Hug, Alexis G. Grimaldi, Kai Kruse and Juan M. Vaquerizas. Chromatin architecture emerges during zygotic genome activation independent of transcription. Cell 169: 216-228, April 6th, 2017, DOI: 10.1016/j.cell.2017.03.024

Journal reference: Cell

Provided by: Max Planck Society

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