C2c2: We can now 'cut and paste' RNA in addition to DNA, and it could disable viruses

Juan Gaertner/Shutterstock.com

Science gets smarter.

You've probably heard of CRISPR - the gene editing tool that essentially lets scientists cut and paste DNA, removing things like HIV and muscular dystrophyf rom our cells - and now scientists have discovered a way to edit RNA with just as much precision.

RNA is DNA's close biological cousin, responsible for translating messages from the nucleus to the rest of the cell, and being able to change it could open up all-new disease-fighting possibilities.

Just like CRISPR/Cas9 editing, the new procedure selectively cuts up RNA, which gives us microscopic control over genetic information, and the researchers behind it say it could open up the method could be used to block viruses and halt disease in its tracks.

"RNA is the blueprint through which genes regulate cellular processes," biologist Oliver Rackham at the University of Western Australia, who wasn't involved in the study, told Colin Barras from New Scientist. "The exciting thing about this study is that it now opens up the RNA world to the ease of experimental design afforded by CRISPR."

While DNA carries the genetic code for our bodies, RNA carries out those instructions and puts that code to work. Basically, DNA has the recipes but RNA reads that recipe and directs all the cooking.

And while scientists used to think the RNA was just a 'middleman', they've realised over the past few decades that it does so much more than that - it can control which proteins are produced in the cell.

In fact, the original CRISPR/Cas9 method uses a combination of DNA and RNA information to target viruses in the body, and identify genetic code that needs replacing. But right now, it focusses solely on DNA.

Now an international team of scientists, led by Feng Zhang from the Broad Institute of MIT and Harvard, has discovered another version of CRISPR called C2c2, and it can be used to slice up RNA molecules exclusively.

Just like CRISPR/Cas9, the C2c2 system was discovered inside bacteria, where it works like a pair of molecular scissors to 'cut out' any invading pathogen's RNA in order to disable an attack.

But the difference is that C2c2 only targets RNA, not DNA itself, which means scientists can use it to make changes and control what happens within a cell, without changing the genetic code itself.

Zhang's team tested the system by inserting C2c2 into E. coli bacteria, and showed they were able to use it to silence a gene simply by targeting RNA. This is because they attacked the RNA carrying the instructions from the gene, so the gene was effectively turned off, without the DNA being touched.

The researchers say C2c2 should also be able to bind to RNA without cutting it up, which would allow scientists to track the movement of RNA in a cell and monitor the effect that changing genetic information has on a single cell, or throughout the whole body.

Whereas DNA changes are permanent, RNA changes could be tweaked as treatment progresses, which gives a lot more opportunity for fighting disease.

"C2c2 opens the door to an entirely new frontier of powerful CRISPR tools," said Zhang. "There are an immense number of possibilities for C2c2, and we are excited to develop it into a platform for life science research and medicine."

While the technique did produce some collateral damage in this study - some RNA that looked similar to the target molecules was unintentionally attacked - it's still early days, and Rackham told New Scientist that the feature could actually be useful in some treatments.

"In a cancer treatment, for example, wiping out 'bystander' RNA as well as target RNA might offer a more effective way to fight a tumour," reports Barras.

This isn't the first time scientists have been able to target RNA instead of DNA through gene-editing techniques, but the difference is C2c2 uses a more natural and more versatile approach, and potentially has a range of different uses. We can't wait to see what scientists do with it.

The results of the study have been published in Science.

ORIGINAL: Science Alert
DAVID NIELD
3 JUN 2016

Bitter fight over CRISPR patent heats up

Unusual battle among academic institutions holds key to gene-editing tool’s future use.

Steve Jennings/Getty/Breakthrough Prize.Biologist Jennifer Doudna of the University of California, Berkeley, helped to develop the CRISPR gene-editing system.

A versatile technique for editing genomes has been called the biggest biotechnology advancesince the polymerase chain reaction (PCR), and the US Patent and Trademark Office (USPTO) is set to determine who will reap the rewards.

On 11 January, the USPTO granted a request to review a key patent awarded for the technique, known as CRISPR–Cas9. The outcome of the ensuing proceedings, called a patent interference, could be worth millions to the research institutions that are at war over the relevant patents. It might also influence who is allowed to use the technology — and under what terms.

“This is an absolutely humungous biotech patent dispute,” says legal scholar Jacob Sherkow of New York Law School. “We’re all waiting with bated breath.”

CRISPR–Cas9 is a bacterial defence system that uses the enzyme Cas9 to snip DNA at sites determined by the sequence of a ‘guide’ strand of RNA. Scientists can disable, replace or tweak genes by using the technique to rewrite snippets of DNA sequences. Use of the technology in research has exploded, thanks to CRISPR–Cas9’s relative simplicity and versatility compared to other gene-editing methods. Several companies have sprung up to harness the technique for generating improved crops, research reagents and therapies for human genetic diseases.

The roots of the CRISPR–Cas9 dispute date back to 2012, when researchers reported that they had reprogrammed the system to cut strands of isolated DNA at sites of their choosing1. The team, led by biologists Jennifer Doudna at the University of California, Berkeley, and Emmanuelle Charpentier, now at the Max Planck Institute for Infection Biology in Berlin and Umeå University in Sweden, filed a patent application on 15 March 2013.

By then, publications had emerged from other groups showing that the method works in human cells2–4 and bolstering dreams of CRISPR-based gene therapies — the basis for several companies that have sprung up to capitalize on the technique.

One of those groups, led by synthetic biologist Feng Zhang of the Broad Institute and the Massachusetts Institute of Technology, both in Cambridge, filed a patent application for the CRISPR–Cas9 technique in October 2013. The institutions filed the patent under a special expedited review programme, and it was granted in April 2014. Zhang has since been awarded additional patents on the technology. The original Doudna–Charpentier patent remains under review.

A pitched battle

In April 2015, the Berkeley team asked the USPTO to begin an interference proceeding to determine which team was the first to invent the technique. The proceedings will be much like a court case, with both sides presenting evidence culled from publications and laboratory notebooks. “Once the [USPTO] declares an interference, that’s really when the fur is going to fly,” Sherkow predicted in a June interview.

Nature special:CRISPR — the good, the bad and the unknown

The patent interference is also a testament to the high stakes involved: companies aiming to use CRISPR–Cas9 for gene therapy have raised hundreds of millions in venture capital and other funds in under three years. One company, Editas Medicine in Cambridge, Massachusetts, has already filed to go public.

Arti Rai, a legal scholar at Duke University in Durham, North Carolina, says that it is unusual for academic research institutions to battle so intensely over a patent. Instead, such institutions usually come to an agreement to share rights to the invention. “This seems more bitter than disputes I’ve heard of in the past,” she adds.

The two patents in question make broad claims to 'foundational' intellectual property thought to be necessary for most lucrative CRISPR–Cas9 applications. But many patents have been filed on CRISPR–Cas9 technologies, and there is still the chance that the winner of the interference will face additional challenges in court. Zhang's group has also reported another enzyme, called Cpf1, that could provide an alternative to Cas9. Researchers expect other alternatives to emerge with time.

As for the various CRISPR–Cas9 companies, Zhang remains involved in Editas, which was founded by both Zhang and Doudna, among others, in 2013. Doudna has since severed ties with Editas and thrown her support behind Intellia Therapeutics, also in Cambridge. Charpentier, meanwhile, co-founded CRISPR Therapeutics of Basel, Switzerland.

Licensing looms

For now, it is unclear how the dispute will affect researchers who use CRISPR–Cas9, if it does so at all. Academics who might use the technology for basic research make unattractive targets for patent lawsuits, says Rodney Sparks, a biotechnology patent counsel at the University of Virginia in Charlottesville. “Patent holders might send out a few cease-and-desist letters, but they probably won’t sue academic researchers,” he says. Doing so would take time and money with little reward: the spoils in a patent lawsuit are typically damages or a share of royalties from a marketed product. That leaves little to gain from suing academics who are not selling anything. But those who intend to use their research as the basis for a start-up company will need to be wary, Sparks says.

Some patent holders do ask that even scientists doing basic research take out a licence on a patented technology, typically for a fairly small fee. Such was the case for PCR, says Warren Woessner, a lawyer at Schwegman Lundberg and Woessner in Minneapolis, Minnesota. Woessner recalls how, during his previous career as a scientist, his institution decided to patent a method he developed. Officials at the institution later noticed that someone had published a paper that used the technique without a licence. “They sent the professor a little note,” recalls Woessner.“‘We have a patent on this. Pay up.’” The professor did.

The Broad Institute has noted on its website that it will continue to make CRISPR–Cas9 reagents available to the community, and has given no indication that it will pursue licensing fees from academics. But Sherkow warns against assuming that the spirit of academic camaraderie will prevail: licensing revenue has become increasingly important, particularly for major research institutions, he says. “We’re just living in a brave new world these days.”
Nature doi:10.1038/nature.2015.17961

ORIGINAL: Nature

Heidi Ledford

12 January 2016

MIT, Broad scientists overcome key CRISPR-Cas9 genome editing hurdle

Courtesy of Ian Slaymaker/Broad Institute of MIT and Harvard

The researchers used structural knowledge of Cas9 to guide engineering of a highly specific genome-editing tool.

MIT, Broad scientists overcome key CRISPR-Cas9 genome editing hurdle

Team re-engineers system to dramatically cut down on editing errors; improvements advance future human applications.

The following is adapted from a press release issued today by the Broad Institute.

Researchers at the Broad Institute of MIT and Harvard and the McGovern Institute for Brain Research at MIT have engineered changes to the revolutionary CRISPR-Cas9 genome editing system that significantly cut down on “off-target” editing errors. The refined technique addresses one of the major technical issues in the use of genome editing.

The CRISPR-Cas9 system works by making a precisely targeted modification in a cell's DNA. The protein Cas9 alters the DNA at a location that is specified by a short RNA whose sequence matches that of the target site. While Cas9 is known to be highly efficient at cutting its target site, a major drawback of the system has been that, once inside a cell, it can bind to and cut additional sites that are not targeted. This has the potential to produce undesired edits that can alter gene expression or knock a gene out entirely, which might lead to the development of cancer or other problems. 

In a paper published today in Science, Feng Zhang and his colleagues report that changing three of the approximately 1,400 amino acids that make up the Cas9 enzyme from S. pyogenes dramatically reduced “off-target editing” to undetectable levels in the specific cases examined. Zhang is the W.M. Keck Career Development Professor in Biomedical Engineering in MIT’s departments of Brain and Cognitive Sciences and Biological Engineering, and a member of both the Broad Institute and McGovern Institute.

Zhang and his colleagues used knowledge about the structure of the Cas9 protein to decrease off-target cutting. DNA, which is negatively charged, binds to a groove in the Cas9 protein that is positively charged. Knowing the structure, the scientists were able to predict that replacing some of the positively charged amino acids with neutral ones would decrease the binding of “off target” sequences much more than “on target” sequences.

After experimenting with various possible changes, Zhang’s team found that mutations in three amino acids dramatically reduced “off-target” cuts. For the guide RNAs tested, “off-target” cutting was so low as to be undetectable.

The newly-engineered enzyme, which the team calls “enhanced” S. pyogenes Cas9, or eSpCas9, will be useful for genome editing applications that require a high level of specificity. The Zhang Lab is immediately making the eSpCas9 enzyme available for researchers worldwide. The team believes the same charge-changing approach will work with other recently described RNA-guided DNA targeting enzymes, including Cpf1, C2C1, and C2C3, which Zhang and his collaborators reported on earlier this year.

The prospect of rapid and efficient genome editing raises many ethical and societal concerns, says Zhang, who is speaking this morning at the International Summit on Gene Editing in Washington. “Many of the safety concerns are related to off-target effects,” he says. “We hope the development of eSpCas9 will help address some of those concerns, but we certainly don’t see this as a magic bullet. The field is advancing at a rapid pace, and there is still a lot to learn before we can consider applying this technology for clinical use.”

ORIGINAL: MIT News

News Office 

December 1, 2015

Can We Identify Every Kind of Cell in the Body?

A microscopic quest to find out what we’re really made of.

WHY IT MATTERS

There is still no accurate atlas of human cell types.

A microfluidic device (at center) can carry out experiments on individual cells.

How many types of cells are there in the human body? Textbooks say a couple of hundred. But the true number is undoubtedly far larger.

Aviv Regev. Regev received her M.Sc. from Tel Aviv University, studying biology, computer science, and mathematics in the Interdisciplinary Program for the Fostering of Excellence. She received her Ph.D. in computational biology from Tel Aviv University. Photo: Broad Institute

Piece by piece, a new, more detailed catalogue of cell types is emerging from labs like that of Aviv Regev at the Broad Institute, in Cambridge, Massachusetts, which are applying recent advances in single-cell genomics to study individual cells at a speed and scale previously unthinkable.

The technology applied at the Broad uses fluidic systems to separate cells on microscopic conveyor belts and then submits them to detailed genetic analysis, at the rate of thousands per day. Scientists expect such technologies to find use in medical applications where small differences between cells have big consequences, including cell-based drug screens, stem-cell research, cancer treatment, and basic studies of how tissues develop.

Regev says she has been working with the new methods to classify cells in mouse retinas and human brain tumors, and she is finding cell types never seen before. “We don’t really know what we’re made of,” she says.

Other labs are racing to produce their own surveys and improve the underlying technology. Today a team led by Stephen Quake of Stanford University published its own survey of 466 individual brain cells, calling it “a first step” toward a comprehensive cellular atlas of the human brain.

Such surveys have only recently become possible, scientists say. “A couple of years ago, the challenge was to get any useful data from single cells,” says Sten Linnarsson, a single-cell biologist at the Karolinska Institute in Stockholm, Sweden. In March, Linnarsson’s group used the new techniques to map several thousand cells from a mouse’s brain, identifying 47 kinds, including some subtypes never seen before.

Historically, the best way to study a single cell was to look at it through a microscope. In cancer hospitals, that’s how pathologists decide if cells are cancerous or not: they stain them with dyes, some first introduced in the early 1900s, and consider their location and appearance. Current methods distinguish about 300 different types, says Richard Conroy, a research official at the National Institutes of Health.

Individual cells are captured and separated in bubbles of liquid, readying them for analysis.

The new technology works instead by cataloguing messenger RNA molecules inside a cell. These messages are the genetic material the nucleus sends out to make proteins. Linnarsson’s method attaches a unique molecular bar code to every RNA molecule in each cell. The result is a gene expression profile, amounting to a fingerprint of a cell that reflects its molecular activity rather than what it looks like.

“Previously, cells were defined by one or two markers,” says Linnarsson. “Now we can say what is the full complement of genes expressed in those cells.”

Although researchers determined how to accurately sequence RNA from a single cell a few years ago, it’s only more recently that clever innovations in chemistry and microfluidics have led to an explosion of data. A California company, Cellular Research, showed this year that it could sort cells into micro-wells and then measure the RNA of 3,000 separate cells at once, at the cost of few pennies a cell.

Scientists think the new single-cell methods could overturn previous research findings. That is because previous gene expression studies were based on tissue samples or blood specimens containing thousands, even millions, of cells. Studying such blended mixtures meant researchers were seeing averages, says Eric Lander, head of the Broad Institute.

“Single-cell genomics has come of age in an unbelievable way in just the last 18 months,” Lander told an audience at the National Institutes of Health this year. “And once you realize we are at the point of doing individual cells, how could you ever put up with a fruit smoothie? It is just nuts to be doing genomics on smoothies.”

Lander, one of the leaders of the Human Genome Project, says it may be time to turn pilot projects like those Regev is leading into a wider effort to create a definitive atlas—one cataloguing all human cell types by gene activity and tracking them from the embryo all the way to adulthood.

“It’s a little premature to declare a national or international project until there’s been more piloting, but I think it’s an idea that’s very much in the air,” Lander said in a phone interview. “I think [in two years] we’re going to be in the position where it would be crazy not to have this information. If we had a periodic table of the cells, we would be able to figure out, so to speak, the atomic composition of any given sample.”

Gene profiles might eventually be combined with other efforts to study single cells. Paul Allen, Microsoft’s cofounder, said last December he would be spending $100 million to create a new scientific institute, the Allen Institute for Cell Science It will study stem cells and video their behavior under microscopes as they develop into various cell types, with the ultimate goal of creating a massive animated model. Rick Horwitz, who leads that effort, says that it will serve as a kind of Google Earth for exploring a cell’s life cycle.

The eventual payoff of collecting all this data, says Garry Nolan, an immunologist at Stanford University, won’t be just a catalogue of cell types, but a deeper understanding of how cells work together. “The single-cell approach is a way station that needs to be understood on the way to understanding the greater system,” he says. “In 50 years, we’ll probably be measuring every molecule in the cell dynamically.”

ORIGINAL: MIT Tech Review

By Courtney Humphries

May 18, 2015

Prostate cancer’s complexities unveiled through whole genome sequencing

ORIGINAL: Broad Institute
By Haley Bridger, Broad Communications, 

February 9th, 2011

Genomic rearrangements occur when pieces of chromosomes break off and reattach in the wrong places. This can generate chimeric chromosomes, depicted here as different colored balls of DNA yarn. Researchers found a surprisingly large number of complex rearrangements in prostate cancer. Image by Sigrid Knemeyer

Fifty years ago, Philadelphia researchers peered through a microscope at cells from a patient with leukemia and made a startling discovery: they saw an abnormally shortened chromosome, the result of two chromosomal pieces breaking off and swapping places in these cancer cells. The sighting of this shuffling – known as a genomic rearrangement or translocation – would lead to a landmark cancer drug tailored to patients whose tumors harbored this genomic alteration.

This week, a research team led by scientists at the Broad Institute, Dana-Farber Cancer Institute and Cornell Weill College of Medicine report in Nature that they have uncovered an even more complex series of such translocations in prostate cancer, a discovery revealed not through the lens of a microscope but through a technique known as whole genome sequencing. These rearrangements in turn have cast a spotlight on genes that may be playing an important role in prostate cancer.

“Many of these features were invisible before,” said co-senior author Levi Garraway, a senior associate member of the Broad Institute and a medical oncologist and assistant professor at the Dana–Farber and Harvard Medical School. “Now, we’re realizing that by sequencing whole genomes in prostate cancer, there’s a lot more to see. These discoveries are teaching us a great deal about prostate cancer biology that we simply hadn’t appreciated previously.”

Prostate cancer is the second most lethal cancer in American men, responsible for more than 30,000 deaths and more than 200,000 new cases each year. A major goal of prostate cancer research is to identify potential drug targets as well as genetic characteristics within tumors that could distinguish indolent and aggressive forms of the disease, and ultimately improve diagnostics and treatment.

Whole genome sequencing enables researchers to look across the entire genome of a tumor to see global changes and patterns. Analyzing and sequencing a full cancer genome is a major technical feat – to date, most whole genome sequencing projects in cancer have examined a single tumor sample. In the prostate cancer study, however, researchers laid bare the full genetic blueprints of seven tumor samples, comparing each to a normal genome from the same person.

“This was the first study in prostate cancer in which whole genome sequencing has been performed on multiple genomes,” said co-first author Michael Berger, who worked on the project as a Cancer Program research scientist. “In dong so, we’ve identified novel aspects of prostate cancer biology.”

Other sequencing methods that target specific sections of the genome allow researchers to see smaller changes, such as point mutations in which one chemical letter of DNA is changed. Imagining the genome as text, these kinds of mutations are like spelling errors. Whole genome sequencing allows scientists to detect these misspellings as well as the rearrangement of whole paragraphs or pages of genomic text.

“One of the big surprises is the fact that prostate cancer doesn’t have a large number of misspellings, but instead has a large, significant number of rearrangements,” said co-senior author Mark Rubin, a genitourinary pathologist and professor in pathology and laboratory medicine at Weill Cornell Medical College. “We would never have guessed that there were so many genomic alterations of this type before now because we didn’t have the right tools to look for them.”

Using this sequencing approach, the researchers were able to see instances where chromosomes have been taken apart and put back together again in the wrong way. “It looks as though a bunch of breaks have been introduced on different chromosomes, and when the DNA repair machinery tried to repair these DNA strands, pieces got swapped, and attached to the wrong partners,” said Michael Lawrence, a co-first author and Broad computational biologist. “In the case of some of these rearrangements, we see a closed cycle of translocation in which no material has been lost – just sort of scrambled.”

This scrambling can lead to the creation of “fusion proteins,” the result of two genes becoming joined together. In the case of the Philadelphia chromosome, the fusion of two genes permanently turns on a protein that signals cells to grow, leading to chronic myelogenous leukemia (CML). Twenty years after this gene fusion was discovered, scientists developed a drug known as imatinib (sometimes called Gleevec), which binds to and thereby deactivates this abnormal protein. The story of this discovery has inspired modern cancer research.

Rubin, who is also a pathologist at NewYork-Presbyterian Hospital/Weill Cornell Medical Center, and his colleagues helped discover another gene fusion – TMPRSS2-ERG – in prostate cancer a few years ago. Just as in the case of the Philadelphia chromosome, TMPRSS2-ERG forms when two chromosomes swap pieces. In the latest study, researchers have found genomic swapping of even greater complexity. Instead of two pieces of genetic material getting swapped, many pieces are involved in a series of interchanges.

These breaks and repairs fall at the sites of some genes known to play a role in cancer. Several tumors contained rearrangements disrupting genes that had not been associated with prostate cancer before, such as the gene that codes for the protein CADM2, part of a family of proteins that prevent tumors from forming (known as “tumor suppressors”). Three samples also contained mutations involving members of the heat shock protein family, molecules that play an important, protective role and keep proteins from losing their proper shape. Anti-cancer drugs that inhibit these proteins are currently in clinical trials, but it is not yet clear whether prostate cancers will be vulnerable to such drugs.

Other recurring genomic rearrangements involve the genes PTEN and MAGI2. PTEN is a well-known tumor suppressor gene and MAGI2 appears to be its helpmate; mutations to one or both genes may set cells on the path toward becoming cancerous. Drugs that inhibit the pathway these genes influence are also being developed, raising the possibility that the drugs could be applied to prostate cancer.

The discovery of these alterations was made possible through the development of new computational tools, the careful collection and selection of tumor samples, and collaborations across institutions and across groups at the Broad. Garraway’s group continues to work closely with Rubin’s to follow up on genes of interest and sequence additional samples. The researchers also received help from many of the Broad’s platforms.

“This whole project was a collaboration that cut across the entire Broad,” said Berger. “The Biological Samples Platform, Genetic Analysis Platform, Genome Sequencing Platform and their informatics group, Cancer Program leadership, and the scientists and project management in the Cancer Program all shared the same vision on this project.”

Other Broad researchers who contributed to this project include Kristian Cibulskis, Andrey Sivachenko, Carrie Sougnez, Robert Onofrio, Scott Carter, Lauren Ambrogio, Timothy Fennell, Melissa Parkin, Gordon Saksena, Douglas Voet, Alex Ramos, Trevor Pugh, Jane Wilkinson, Sheila Fisher, Wendy Winckler, Scott Mahan, Kristin Ardlie, Jennifer Baldwin, Lynda Chin, Stacey Gabriel, Todd Golub, Matthew Meyerson, Eric Lander, and Gad Getz. Researchers from the Weizmann Institute of Science, Yale University, and the Prostate Cancer Foundation also contributed to this work.

Funding for the project was provided by the Prostate Cancer Foundation, the Howard Hughes Medical Institute, the National Human Genome Research Institute, the Kohlberg Foundation, the National Cancer Institute, the National Institutes of Health, the Department of Defense, the Dana-Farber/Harvard Cancer Center Prostate Cancer SPORE grant, and the Starr Cancer Consortium.


Paper(s) cited:

Berger M. et al. The genomic complexity of primary human prostate cancer. Nature. Published online February 9, 2011. DOI:10.1038/nature09744

Researchers identify biochemical functions for most of the human genome

ORIGINAL: MIT News

Anne Trafton, MIT News Office

New map finds genetic regulatory elements account for 80 percent of our DNA.

Only about 1 percent of the human genome contains gene regions that code for proteins, raising the question of what the rest of the DNA is doing. Scientists have now begun to discover the answer: About 80 percent of the genome is biochemically active, and likely involved in regulating the expression of nearby genes, according to a study from a large international team of researchers.

The consortium, known as ENCODE (which stands for “Encyclopedia of DNA Elements”), includes hundreds of scientists from several dozen labs around the world. Using genetic sequencing data from 140 types of cells, the researchers were able to identify thousands of DNA regions that help fine-tune genes’ activity and influence which genes are expressed in different kinds of cells.

Just as the sequencing of the human genome helped scientists learn how mutations in protein-coding genes can lead to disease, the new map of noncoding regions should provide some answers on how mutations in the regulatory elements lead to diseases such as lupus and diabetes, says Manolis Kellis, an associate professor of computer science at MIT, an associate member of the Broad Institute and an author of a paper describing the findings in the Sept. 5 online edition of Nature.

“Humans are 99.9 percent identical to each other, and you only have one difference in every 300 to 1,000 nucleotides,” Kellis says. “What ENCODE allows you to do is provide an annotation of what each nucleotide of the genome does, so that when it’s mutated, we can make some predictions about the consequences of the mutation.”

Kellis, who leads MIT’s Computational Biology Group, is one of the principal investigators involved in the Nature paper. The ENCODE collaboration is publishing about two dozen additional papers this week detailing the new results.

Mapping noncoding DNA

ENCODE was established in 2003 to extend our understanding of the human genome beyond protein-coding genes. One way to do that is by studying the chemical modifications of individual stretches of DNA, which control when genetic regions will be active. These modifications vary by cell type and can modify either DNA directly or the histone proteins that DNA wraps around.

To map these modifications, known collectively as the epigenome, the research groups had to collect many different kinds of data from different cell types. Some labs measured DNA or histone modifications, while others gauged the accessibility of different stretches of DNA by cutting it into fragments with enzymes. 

Kellis and his group were among the computational scientists leading the effort to analyze and integrate the huge amount of data generated by different labs. “Given that we were getting more than 1,000 data sets, we had to figure out ways to automatically calibrate experiments,” says Anshul Kundaje, a research scientist in MIT’s Computational Biology Group. “We developed an almost purely automated system that did all of this.”

The ENCODE researchers found that 80 percent of the genome experiences some kind of biochemical event, such as binding to proteins that regulate how often a neighboring gene is utilized. They also discovered that the same regulatory region can play different roles, depending on what type of cell it’s acting in.

The findings should have a major impact on scientists’ understanding of human biology and how genomic variations can cause disease, says Ben Raphael, an associate professor of computer science at Brown University.

“The most exciting part is now we’re getting a whole genome annotation of functional elements,” says Raphael, who was not part of the research team. “Every time you want to understand what a particular piece of the genome is doing, you can use the data from this project.”

Human variation

The researchers also studied the conservation of nucleotides — the A, T, C and G “letters” of DNA — in the newly identified regulatory regions. Nucleotides are conserved if they remain the same over long evolutionary periods, which can be measured by analyzing the variability between species, or among individuals within a species. 

A recent paper by Kellis and colleagues showed that 5 percent of noncoding DNA is conserved across mammals. In one of the ENCODE companion papers appearing online Sept. 5 in Science, Kellis and MIT postdoc Lucas Ward show that an additional 4 percent is conserved within the human lineage, suggesting that those elements control recently evolved traits, some of which are unique to humans.

When the researchers looked at the functions of genes near newly evolved regulatory regions, they found many genes that encode regulators that activate other genes. “Genes involved in the nerve growth pathway and color vision, both of which have been hypothesized to be recent innovations in the primate lineage, are enriched in human-constrained elements in non-conserved regions,” Ward says.

The researchers found that the most highly conserved nucleotides were also the ones most likely to be associated with disease when mutated. They also showed that variants associated with autoimmune diseases such as lupus and rheumatoid arthritis are located in regions active only in immune cells, while variants linked to metabolic diseases are in regions active only in liver cells.

In their next phase, the ENCODE researchers hope to determine just how those variations lead to human disease. 

“What we’ve done over this series of papers is effectively paint a set of reference annotations of common human genome function,” Kellis says. “Our next steps will be to personalize these maps — to basically ask how they vary naturally between individuals, by profiling different cell types from different people, and how their variation relates to human disease and complex human traits.”

In one follow-up project, Kellis and colleagues are comparing activity levels of regulatory elements in different cell types from the same person, across many individuals. Another project is looking at DNA modification patterns across the entire genome of many individuals, in hopes of identifying how variation of specific elements relates to disease.

The research was funded by the National Human Genome Research Institute.