From UNC: “The Cure Code”

University of North Carolina

July 18th, 2017
Alyssa LaFaro

Davis can now walk fully unsupported and live a relatively normal life thanks to a correct diagnosis from UNC researchers within the NCGENES project. No image credit.

“Consider this: In 1969, if a disease-linked gene was found in humans, scientists had no simple means to understand the nature of the mutation, no mechanism to compare the altered gene to normal form, and no obvious method to reconstruct the gene mutation in a different organism to study its function. By 1979, that same gene could be shuttled into bacteria, spliced into a viral vector, delivered into the genome of a mammalian cell, cloned, sequenced, and compared to the normal form.” —Siddhartha Mukherjee, “The Gene: An Intimate History”

“I can move my toes,” Elizabeth Davis says.

Her 9-year-old son looks at her in awe. The two stand, wide-eyed in the middle of a Verizon Wireless store in Goldsboro, North Carolina. Davis leans hard against her crutches, staring at her feet. She looks up and smiles.

At age 37 — for the first time in 31 years — Davis can uncurl her toes from a locked position, the symptom of a condition gone misdiagnosed for just as long. Three months later, she sheds her crutches, walking fully unsupported — something she hasn’t done since she was 14 years old.

In 1975, the same year Davis was born, UNC microbiologists Clyde Hutchison and Marshall Edgell experienced a different kind of life-changing event. They’d been working rigorously to isolate DNA within the smallest-known virus at the time, Phi-X174. More than anything, they wanted to understand how to read the genetic code. Then, later that year and across the pond at St. John’s College in Cambridge, Fred Sanger figured it out. The British biochemist became the first person to develop a relatively rapid method for sequencing DNA, a discovery that won him a Nobel Prize in Chemistry — for the second time.

In response to Sanger’s discovery, Hutchison took a sabbatical and headed to England to work in his lab. During his first year there, he helped uncover the entire sequence of Phi-X174 — the first time this had been done for any organism. While there, he realized the new ability to read DNA could help him and Edgell solve a different problem they’d been having back in North Carolina: fusing two pieces of DNA code together to create an entirely different sequence.

After returning to Chapel Hill, Hutchison continued his work with Edgell and also Michael Smith, a researcher at the University of British Columbia who he met while working in Sanger’s lab. Together, the trio successfully fused two differing DNA strands using a more flexible approach to site-directed mutagenesis — a technique that makes gene therapy possible today. They published their results in 1978. Smith would go on to receive the Nobel Prize for this work in 1992.

—-

The scientific breakthroughs of the 1970s changed the field of genetics forever. In 1980, Sanger received the Nobel Prize for Chemistry for his contributions, along with Walter Gilbert (Harvard), who discovered that individual modules from different genes mix and match to construct entirely new genes; and Paul Berg (Stanford), who developed a technique for splicing recombinant DNA.

Meanwhile, researchers in Chapel Hill continued to chip away at the mysteries of the gene. Oliver Smithies, who came to UNC in 1987, would later win the Nobel Prize for his work in gene targeting using mouse models. That same year, UNC cancer geneticist Michael Swift and team discover the AT gene, which predisposes women to breast cancer; and George McCoy becomes the first clinical trial participant in the world to receive the genetically engineered Factor VIII gene to treat his hemophilia at the then UNC-Thrombosis and Hemostasis Center.

Genetics was changing the world. And this was only the beginning.

An unsolved mystery

One year after Sanger won the Nobel Prize, Elizabeth Davis turned 6. She soon began walking on her toes, which had suddenly, one day, curled under in pain, making it nearly impossible for her to stride with feet flat on the ground. Her knees knocked together as she struggled to move with the swift pace characteristic of a child her age. Davis continued to walk on her toes for years.

“I would even brace the school walls when walking down the hallway,” she says. Eventually, the pain became unbearable. By the time she was 12, she’d resigned herself to crutches.

Doctors believed Davis’ condition could be treated with foot surgery, misdiagnosing her condition for years. By age 14, she had already undergone three procedures — two to lengthen her Achilles tendons and an experimental bone fusion. But each surgery offered little to no relief, and walking only grew more painful for Davis, both physically and emotionally. As her condition worsened, her classmates became cruel — so much so that she dropped out of high school when she was just 16.

By age 20, Davis grew restless. “The pain was constant,” she remembers. “I could hardly move my legs — they just felt weak. I would drag them behind me as I used my crutches. I couldn’t even lift them.” Doctors suggested she undergo a third Achilles tendon lengthening surgery, the result of which minimally improved her condition.

“By that age, I just wanted more,” Davis says. “I just wanted to do things, to go places. I wanted the surgery to work. But it didn’t. And the pain continued.”

It would be another 17 years before doctors realized the problem was hidden in her genome.

The birth of a department

In 1990, the start of the Human Genome Project — an international research program to map out the 20,000 genes that define human beings — further fueled new discoveries in the field of genetics. So when Jeff Houpt, then-UNC School of Medicine dean, formed a research advisory committee in 1997 and asked his faculty what the number-one research program the university needed to focus on, they responded: genetics and genome sciences.

Great minds think alike. At the same time, the College of Arts and Sciences was also hosting its own committee that vied to develop a genetics department. “At this point, I had a vision for a pan-university program,” Houpt shares. “This wasn’t just going to be a program of the medical school.”

Along with the College, the schools of public health, dentistry, pharmacy, nursing, and information and library science all wanted in, offering financial assistance to the program. Then-Provost Robert Shelton and Chancellor James Moeser both signed off on it as well. “What we wanted from Shelton and Moeser was more money and more positions,” Houpt remembers. “And they agreed to that.”

By 2000, a hiring committee was ready to interview candidates to chair the new department and genomics center. Terry Magnuson quickly emerged as the lead candidate. He and his team had spent the past 16 years researching developmental abnormalities using genetics and mouse models, successfully changing the genetic background of a mutated gene.

“It was obvious he was going to have a following,” Houpt remembers. “People were going to listen to him because he’s a good scientist. But more than that, it was pretty clear that Terry was interested in building a program, and this university-wide effort appealed to him.”

Unanswered pain

By the time she reached her 30s, Davis’ condition had spread to her arms. She underwent multiple MRIs, nerve and muscle testing, and a spinal tap. She even endured a fifth, unsuccessful surgery on her feet. Physicians misdiagnosed her yet again. A few believed she suffered from hereditary spastic paraplegia, a genetic condition that causes weakness in the legs and hips. Another told her she had cerebral palsy. “But I didn’t want to believe him,” she says — and it’s a good thing she didn’t.

As Davis continued her search for answers, walking grew more and more painful. “I was always in pain,” she admits. “But some weeks were really, really bad — to the point where I couldn’t even move.” She finally succumbed to the assistance of a wheelchair. “I hated it so much. I barely went anywhere.” And when she did, she needed help.

Her mother assisted her regularly with everyday tasks like grocery shopping. Her youngest son, Alex, learned to expertly navigate her around high school gyms, baseball fields, and the local YMCA pool so she could watch her other son, Myles, compete in the plethora of sports he participated in.

“Myles really experienced the worst of it,” Davis says. “I remember one time, in particular. I was taking a shower and knew I was about to fall. I called for him and he came running. He was always there to pick me back up.”

Sequences and algorithms

After the Human Genome Project published its results in 2004, genomic sequencing became an option for people with undiagnosed diseases. But analyzing and understanding the 3 billion base pairs that make up a person’s genetic identity was an expensive process. As time progressed and technology improved, though, the technique became more manageable for both physicians and patients.

Using these new genomic technologies for outpatient care intrigued UNC geneticists James Evans and Jonathan Berg. In 2009, after gathering enough preliminary data, the NIH granted the team the funds to start the North Carolina Clinical Genomic Evaluation by NextGen Exome Sequencing (NCGENES), which uses whole exome sequencing (WES) to uncover the root cause of undiagnosed diseases. Using just two tablespoons of blood, WES tests 1 percent of the genome — a feat that is both miraculous and controversial, creating a whole new wave of ethical questions.

Simply put: “Some people want information that other people don’t,” Evans explains. Most people want to know about genetic disorders that have treatment options, but when it comes to those that don’t, they’d rather not hear it. “Navigating those different viewpoints can be a challenge,” he says. Privacy and confidentiality also present problems within the insurance world. Although protections exist in the realm of medical insurance, major genetic predispositions could have large implications for life, disability, and long-term care insurance.

Today, upward of 50 researchers from across Carolina participate in NCGENES to study everything from the protection of data to the delivery of results. More than 750 people with undiagnosed diseases have undergone testing.

NCGENES wouldn’t exist without the technical infrastructure that tracks, categorizes, and helps analyze genetic material as it makes its way through multiple laboratories — all of which is provided by UNC’s Renaissance Computing Institute (RENCI). A developer of data science cyberinfrastructure, RENCI provides the software programming that helps the team at NCGENES analyze genomes more effectively.

“You need new computer algorithms to solve new science problems,” RENCI Director Stan Ahalt says. “It takes a multidisciplinary team to understand science problems like genetics — and computer code to make that process go fast.”

A transformative experience

By 2013, Davis was in desperate need of a new algorithm. Thankfully, that year, she was referred to Jane Fan, a pediatric neurologist at UNC. After studying Davis’ file, Fan felt sure that the doctors who tried to diagnose her condition failed, making her the perfect candidate for NCGENES.

Four tubes of blood, 100,000 possible genetic locations, and just over six months later, Fan called Davis. A single gene mutation called GTPCH1 impairs her ability to produce dopa, an amino acid crucial for nervous system function. “I had to hear it in person before I believed it,” Davis admits. “I had been misdiagnosed many times before.”

Not only were UNC geneticist James Evans and his NCGENES team finally able to accurately diagnose Davis, but they were able to treat it — with something as simple as a pill. A pill that has been on the market since 1988, used to treat patients with Parkinson’s disease.

And just like that, Davis ‘life was changed forever by genome sequencing.

Three days after she took one-quarter of a pill, movement returned to her toes while standing in the middle of a Verizon Wireless store in Goldsboro. She began to cry.

Top-five in the country

UNC’s genetics department has ranked in the top-five programs for NIH funding across the nation every year since 2012 (and top-10 each year since 2006). “I think we’ve built one of the best genetics departments in the country,” Magnuson says. In 2016 alone, genetics department faculty brought $38 million to Carolina.

Houpt agrees with Magnuson’s sentiment. “The genetics department is a great example of how universities should run,” he says. “People need to put aside their own interests and see what’s needed. Terry is a leader who’s made each school involved feel like it’s their program and not just a medical school program – which is why he’s now the vice chancellor for research.”

Today, more than 80 faculty members from across campus conduct world-recognized genetics research in multiple disciplines.

Ned Sharpless, for example, focuses on cancer. Most recently, the director of the UNC Lineberger Comprehensive Cancer Center lead a study that paired UNCseq — a genetic sequencing protocol that produces volumes of genetic information from a patient’s tumor — with IBM Watson’s ability to quickly pull information from millions of medical papers. A procedure much too intense and time-consuming for the human mind, this data analysis can help physicians make more informed decisions about patient care.

Another member of Carolina’s Cancer Genetics Program, Charles Perou uses genomics to characterize the diversity of breast cancer tumors — research that helps doctors guarantee patients more individualized care. In 2011, he cofounded GeneCentric, which uses personalized molecular diagnostic assays and targeted drug development to treat cancer.

In 2015, geneticist Aravind Asokan started StrideBio with University of Florida biochemist Mavis Agbandje-McKenna. The gene therapy company develops novel adeno-associated viral (AAV) vector technologies for treating rare diseases. Although still in its infancy, the company has already partnered with CRISPR Therapeutics and received an initial investment from Hatteras Venture Partners. Asokan has spent nearly a decade studying AAV — and even helped to, previously, cofound Bamboo Therapeutics, acquired by Pfizer for $645 million just last year.

In 2016, current genetics department Chair Fernando Pardo-Manuel de Villena challenged both Darwin’s theory of natural selection and Mendel’s law of segregation through researching a mouse gene called R2d2. In doing so, he found that a selfish gene can become fixed in a population of organisms while, at the same time, being detrimental to “reproductive fitness” — a discovery that shows the swiftness at which the genome can change, creating implications for an array of fields from basic biology to agriculture and human health.

A former student of Oliver Smithies, Beverly Koller uses gene targeting in mice to better understand diseases like cystic fibrosis, asthma, and arthritis — research that will ultimately lead to better treatments. Similarly, Mark Heise observes mice to study diseases caused by viruses including infectious arthritis and encephalitis (inflammation of the brain). Both researchers are part of the Collaborative Cross project, a large panel of inbred mouse strains that help map genetic traits — a resource that is UNC lead, according to Magnuson.

Genetics research stems far beyond the UNC School of Medicine. In 2009, for example, chemist Kevin Weeks and his research team decoded the HIV genome, advancing the development of new therapies and treatments. UNC sociologist Gail Henderson runs the Center for Genomics and Society, which provides research and training on ethical, legal, and social implications of genomic research. In 2015, UNC Eshelman School of Pharmacy Dean Bob Blouin helped the school become the first U.S. hub to join the international Structural Genomics Consortium — focused on discovering selective, small molecules and protein kinases to help speed the creation of new medicines for patients.

From crutches to a 5K

After just three months of treatment, Davis walked fully unsupported for the first time since she was 6 years old. She’s since traversed Hershey Park in Pennsylvania, strolled around the World Trade Center in New York, and regularly participated in yoga and spin classes. This past May, she walked her first 5K. “I have crazy endurance,” she says. “When your body feels good, you just want to keep on going.”

Perhaps, more importantly, Davis is able attend Alex’s sports games without assistance. “When I used to walk into the gym on crutches to watch my oldest son play basketball, everyone would look at my crutches and my legs,” she says. “Now, when I go watch my youngest son play, I have so much more confidence walking in to the gym. People see me.”

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Carolina’s vibrant people and programs attest to the University’s long-standing place among leaders in higher education since it was chartered in 1789 and opened its doors for students in 1795 as the nation’s first public university. Situated in the beautiful college town of Chapel Hill, N.C., UNC has earned a reputation as one of the best universities in the world. Carolina prides itself on a strong, diverse student body, academic opportunities not found anywhere else, and a value unmatched by any public university in the nation.

From HMS: “Bringing CRISPR into Focus”

Harvard University

Harvard Medical School

June 29, 2017
KEVIN JIANG

CRISPR-Cas3 is a subtype of the CRISPR-Cas system, a widely adopted molecular tool for precision gene editing in biomedical research. Aspects of its mechanism of action, however, particularly how it searches for its DNA targets, were unclear, and concerns about unintended off-target effects have raised questions about the safety of CRISPR-Cas for treating human diseases.

Harvard Medical School and Cornell University scientists have now generated near-atomic resolution snapshots of CRISPR that reveal key steps in its mechanism of action. The findings, published in Cell on June 29, provide the structural data necessary for efforts to improve the efficiency and accuracy of CRISPR for biomedical applications.

Through cryo-electron microscopy, the researchers describe for the first time the exact chain of events as the CRISPR complex loads target DNA and prepares it for cutting by the Cas3 enzyme. These structures reveal a process with multiple layers of error detection—a molecular redundancy that prevents unintended genomic damage, the researchers say.

High-resolution details of these structures shed light on ways to ensure accuracy and avert off-target effects when using CRISPR for gene editing.

“To solve problems of specificity, we need to understand every step of CRISPR complex formation,” said Maofu Liao, assistant professor of cell biology at Harvard Medical School and co-senior author of the study. “Our study now shows the precise mechanism for how invading DNA is captured by CRISPR, from initial recognition of target DNA and through a process of conformational changes that make DNA accessible for final cleavage by Cas3.”

Target search

Discovered less than a decade ago, CRISPR-Cas is an adaptive defense mechanism that bacteria use to fend off viral invaders. This process involves bacteria capturing snippets of viral DNA, which are then integrated into its genome and which produce short RNA sequences known as crRNA (CRISPR RNA). These crRNA snippets are used to spot “enemy” presence.

Acting like a barcode, crRNA is loaded onto members of the CRISPR family of enzymes, which perform the function of sentries that roam the bacteria and monitor for foreign code. If these riboprotein complexes encounter genetic material that matches its crRNA, they chop up that DNA to render it harmless. CRISPR-Cas subtypes, notably Cas9, can be programmed with synthetic RNA in order to cut genomes at precise locations, allowing researchers to edit genes with unprecedented ease.

To better understand how CRISPR-Cas functions, Liao partnered with Ailong Ke of Cornell University. Their teams focused on type 1 CRISPR, the most common subtype in bacteria, which utilizes a riboprotein complex known as CRISPR Cascade for DNA capture and the enzyme Cas3 for cutting foreign DNA.

Through a combination of biochemical techniques and cryo-electron microscopy, they reconstituted stable Cascade in different functional states, and further generated snapshots of Cascade as it captured and processed DNA at a resolution of up to 3.3 angstroms—or roughly three times the diameter of a carbon atom.

A sample cryo-electron microscope image of CRISPR molecules(left). The research team combined hundreds of thousands of particles into 2D averages (right), before turning them into 3D projections. Image: Xiao et al.

Seeing is believing

In CRISPR-Cas3, crRNA is loaded onto CRISPR Cascade, which searches for a very short DNA sequence known as PAM that indicates the presence of foreign viral DNA.

Liao, Ke and their colleagues discovered that as Cascade detects PAM, it bends DNA at a sharp angle, forcing a small portion of the DNA to unwind. This allows an 11-nucleotide stretch of crRNA to bind with one strand of target DNA, forming a “seed bubble.”

The seed bubble acts as a fail-safe mechanism to check whether the target DNA matches the crRNA. If they match correctly, the bubble is enlarged and the remainder of the crRNA binds with its corresponding target DNA, forming what is known as an “R-loop” structure.

Once the R-loop is completely formed, the CRISPR Cascade complex undergoes a conformational change that locks the DNA into place. It also creates a bulge in the second, non-target strand of DNA, which is run through a separate location on the Cascade complex.

Only when a full R-loop state is formed does the Cas3 enzyme bind and cut the DNA at the bulge created in the non-target DNA strand.

The findings reveal an elaborate redundancy to ensure precision and avoid mistakenly chopping up the bacteria’s own DNA.

CRISPR forms a “seed bubble” state, which acts as an initial fail-safe mechanism to ensure that CRISPR RNA matches its target DNA. Image: Liao Lab/HMS.

“To apply CRISPR in human medicine, we must be sure the system is accurate and that it does not target the wrong genes,” said Ke, who is co-senior author of the study. “Our argument is that the CRISPR-Cas3 subtype has evolved to be a precise system that carries the potential to be a more accurate system to use for gene editing. If there is mistargeting, we know how to manipulate the system because we know the steps involved and where we might need to intervene.”

Setting the sights

Structures of CRISPR Cascade without target DNA and in its post-R-loop conformational states have been described, but this study is the first to reveal the full sequence of events from seed bubble formation to R-loop formation at high resolution.

In contrast to the scalpel-like Cas9, CRISPR-Cas3 acts like a shredder that chews DNA up beyond repair. While CRISPR-Cas3 has, thus far, limited utility for precision gene editing, it is being developed as a tool to combat antibiotic-resistant strains of bacteria. A better understanding of its mechanisms may broaden the range of potential applications for CRISPR-Cas3.

In addition, all CRISPR-Cas subtypes utilize some version of an R-loop formation to detect and prepare target DNA for cleavage. The improved structural understanding of this process can now enable researchers to work toward modifying multiple types of CRISPR-Cas systems to improve their accuracy and reduce the chance of off-target effects in biomedical applications.

“Scientists hypothesized that these states existed but they were lacking the visual proof of their existence,” said co-first author Min Luo, postdoctoral fellow in the Liao lab at HMS. “The main obstacles came from stable biochemical reconstitution of these states and high-resolution structural visualization. Now, seeing really is believing.”

“We’ve found that these steps must occur in a precise order,” Luo said. “Evolutionarily, this mechanism is very stringent and has triple redundancy, to ensure that this complex degrades only invading DNA.”

Additional authors on the study include Yibei Xiao, Robert P. Hayes, Jonathan Kim, Sherwin Ng, and Fang Ding.

This work is supported by National Institutes of Health grants GM 118174 and GM102543.

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Established in 1782, Harvard Medical School began with a handful of students and a faculty of three. The first classes were held in Harvard Hall in Cambridge, long before the school’s iconic quadrangle was built in Boston. With each passing decade, the school’s faculty and trainees amassed knowledge and influence, shaping medicine in the United States and beyond. Some community members—and their accomplishments—have assumed the status of legend. We invite you to access the following resources to explore Harvard Medical School’s rich history.

Harvard is the oldest institution of higher education in the United States, established in 1636 by vote of the Great and General Court of the Massachusetts Bay Colony. It was named after the College’s first benefactor, the young minister John Harvard of Charlestown, who upon his death in 1638 left his library and half his estate to the institution. A statue of John Harvard stands today in front of University Hall in Harvard Yard, and is perhaps the University’s best known landmark.

Harvard University has 12 degree-granting Schools in addition to the Radcliffe Institute for Advanced Study. The University has grown from nine students with a single master to an enrollment of more than 20,000 degree candidates including undergraduate, graduate, and professional students. There are more than 360,000 living alumni in the U.S. and over 190 other countries.

From Science Node: “Cracking the CRISPR clock”

Science Node

05 Jul, 2017
Jan Zverina

SDSC Dell Comet supercomputer

Capturing the motion of gyrating proteins at time intervals up to one thousand times greater than previous efforts, a team led by University of California, San Diego (UCSD) researchers has identified the myriad structural changes that activate and drive CRISPR-Cas9, the innovative gene-splicing technology that’s transforming the field of genetic engineering.

By shedding light on the biophysical details governing the mechanics of CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats) activity, the study provides a fundamental framework for designing a more efficient and accurate genome-splicing technology that doesn’t yield ‘off-target’ DNA breaks currently frustrating the potential of the CRISPR-Cas9- system, particularly for clinical uses.

Shake and bake. Gaussian accelerated molecular dynamics simulations and state-of-the-art supercomputing resources reveal the conformational change of the HNH domain (green) from its inactive to active state. Courtesy Giulia Palermo, McCammon Lab, UC San Diego.

“Although the CRISPR-Cas9 system is rapidly revolutionizing life sciences toward a facile genome editing technology, structural and mechanistic details underlying its function have remained unknown,” says Giulia Palermo, a postdoctoral scholar with the UC San Diego Department of Pharmacology and lead author of the study [PNAS].

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From LBNL: “A Whole-Genome Sequenced Rice Mutant Resource for the Study of Biofuel Feedstocks”

Berkeley Lab

July 5, 2017
Sarah Yang
scyang@lbl.gov
(510) 486-4575

JBEI researchers create open-access web portal to accelerate functional genetic research in plants.

Genome-wide distribution of fast-neutron-induced mutations in the Kitaake rice mutant population (green). Even distribution of mutations is important to achieve saturation of the genome. Colored lines (center) represent translocations of DNA fragments from one chromosome to another. (Credit: Guotian Li and Rashmi Jain/Berkeley Lab).

Rice is a staple food for over half of the world’s population and a model for studies of candidate bioenergy grasses such as sorghum, switchgrass, and Miscanthus. To optimize crops for biofuel production, scientists are seeking to identify genes that control key traits such as yield, resistance to disease, and water use efficiency.

Populations of mutant plants, each one having one or more genes altered, are an important tool for elucidating gene function. With whole-genome sequencing at the single nucleotide level, researchers can infer the functions of the genes by observing the gain or loss of particular traits. But the utility of existing rice mutant collections has been limited by several factors, including the cultivars’ relatively long six-month life cycle and the lack of sequence information for most of the mutant lines.

In a paper published in The Plant Cell, a team led by Pamela Ronald, a professor in the Genome Center and the Department of Plant Pathology at UC Davis and director of Grass Genetics at the Department of Energy’s (DOE’s) Joint BioEnergy Institute (JBEI), with collaborators from UC Davis and the DOE Joint Genome Institute (JGI), reported the first whole-genome sequenced fast-neutron induced mutant population of Kitaake, a model rice variety with a short life cycle.

Kitaake (Oryza sativa L. ssp. japonica) completes its life cycle in just nine weeks and is not sensitive to photoperiod changes. This novel collection will accelerate functional genetic research in rice and other monocots, a type of flowering plant species that includes grasses.

“Some of the most popular rice varieties people use right now only have two generations per year. Kitaake has up to four, which really speeds up functional genomics work,” said Guotian Li, a project scientist at Lawrence Berkeley National Laboratory (Berkeley Lab) and deputy director of Grass Genetics at JBEI.

In a previously published pilot study [Molecular Plant], Li, Mawsheng Chern, and Rashmi Jain, co-first authors on The Plant Cell paper, demonstrated that fast-neutron irradiation produced abundant and diverse mutations in Kitaake, including single base substitutions, deletions, insertions, inversions, translocations, and duplications. Other techniques that have been used to generate rice mutant populations, such as the insertion of gene and chromosome segments and the use of gene editing tools like CRISPR-Cas9, generally produce a single type of mutation, Li noted.

“Fast-neutron irradiation causes different types of mutations and gives different alleles of genes so we really can get something that’s not achievable from other collections,” he said.

Whole-genome sequencing of this mutant population – 1,504 lines in total with 45-fold coverage – allowed the researchers to pinpoint each mutation at a single-nucleotide resolution. They identified 91,513 mutations affecting 32,307 genes, 58 percent of all genes in the roughly 389-megabase rice genome. A high proportion of these were loss-of-function mutations.

Using this mutant collection, the Grass Genetics group identified an inversion affecting a single gene as the causative mutation for the short-grain phenotype in one mutant line with a population containing just 50 plants. In contrast, researchers needed more than 16,000 plants to identify the same gene using the conventional approach.

“This comparison clearly demonstrates the power of the sequenced mutant population for rapid genetic analysis,” said Ronald.

This high-density, high-resolution catalog of mutations, developed with JGI’s help, provides researchers opportunities to discover novel genes and functional elements controlling diverse biological pathways. To facilitate open access to this resource, the Grass Genetics group has established a web portal called KitBase, which allows users to find information related to the mutant collection, including sequence, mutation and phenotypic data for each rice line. Additional information about the database can be found through JGI.

Additional Berkeley Lab scientists who contributed to this work include co-first authors Rashmi Jain and Mawsheng Chern; Tong Wei and Deling Ruan, both affiliated with JBEI’s Feedstocks Division and with Berkeley Lab’s Environmental Genomics and Systems Biology Division; Nikki Pham and Kyle Jones of JBEI’s Feedstocks Division; and Joel Martin, Wendy Schackwitz, Anna Lipzen, Diane Bauer, Yi Peng, and Kerrie Barry of the JGI.

Support for the research at JBEI, a DOE Bioenergy Research Center, and JGI, a DOE Office of Science User Facility, was provided by DOE’s Office of Science. Additional support was provide by the National Institutes of Health and the National Science Foundation.

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A U.S. Department of Energy National Laboratory Operated by the University of California

From HMS: “Bringing CRISPR into Focus”

Harvard University

Harvard Medical School

June 29, 2017
KEVIN JIANG

CRISPR-Cas3 is a subtype of the CRISPR-Cas system, a widely adopted molecular tool for precision gene editing in biomedical research. Aspects of its mechanism of action, however, particularly how it searches for its DNA targets, were unclear, and concerns about unintended off-target effects have raised questions about the safety of CRISPR-Cas for treating human diseases.

Harvard Medical School and Cornell University scientists have now generated near-atomic resolution snapshots of CRISPR that reveal key steps in its mechanism of action. The findings, published in Cell on June 29, provide the structural data necessary for efforts to improve the efficiency and accuracy of CRISPR for biomedical applications.

Through cryo-electron microscopy, the researchers describe for the first time the exact chain of events as the CRISPR complex loads target DNA and prepares it for cutting by the Cas3 enzyme. These structures reveal a process with multiple layers of error detection—a molecular redundancy that prevents unintended genomic damage, the researchers say.

High-resolution details of these structures shed light on ways to ensure accuracy and avert off-target effects when using CRISPR for gene editing.

“To solve problems of specificity, we need to understand every step of CRISPR complex formation,” said Maofu Liao, assistant professor of cell biology at Harvard Medical School and co-senior author of the study. “Our study now shows the precise mechanism for how invading DNA is captured by CRISPR, from initial recognition of target DNA and through a process of conformational changes that make DNA accessible for final cleavage by Cas3.”

Target search

Discovered less than a decade ago, CRISPR-Cas is an adaptive defense mechanism that bacteria use to fend off viral invaders. This process involves bacteria capturing snippets of viral DNA, which are then integrated into its genome and which produce short RNA sequences known as crRNA (CRISPR RNA). These crRNA snippets are used to spot “enemy” presence.

Acting like a barcode, crRNA is loaded onto members of the CRISPR family of enzymes, which perform the function of sentries that roam the bacteria and monitor for foreign code. If these riboprotein complexes encounter genetic material that matches its crRNA, they chop up that DNA to render it harmless. CRISPR-Cas subtypes, notably Cas9, can be programmed with synthetic RNA in order to cut genomes at precise locations, allowing researchers to edit genes with unprecedented ease.

To better understand how CRISPR-Cas functions, Liao partnered with Ailong Ke of Cornell University. Their teams focused on type 1 CRISPR, the most common subtype in bacteria, which utilizes a riboprotein complex known as CRISPR Cascade for DNA capture and the enzyme Cas3 for cutting foreign DNA.

Through a combination of biochemical techniques and cryo-electron microscopy, they reconstituted stable Cascade in different functional states, and further generated snapshots of Cascade as it captured and processed DNA at a resolution of up to 3.3 angstroms—or roughly three times the diameter of a carbon atom.

A sample cryo-electron microscope image of CRISPR molecules(left). The research team combined hundreds of thousands of particles into 2D averages (right), before turning them into 3D projections. Image: Xiao et al.

Seeing is believing

In CRISPR-Cas3, crRNA is loaded onto CRISPR Cascade, which searches for a very short DNA sequence known as PAM that indicates the presence of foreign viral DNA.

Liao, Ke and their colleagues discovered that as Cascade detects PAM, it bends DNA at a sharp angle, forcing a small portion of the DNA to unwind. This allows an 11-nucleotide stretch of crRNA to bind with one strand of target DNA, forming a “seed bubble.”

The seed bubble acts as a fail-safe mechanism to check whether the target DNA matches the crRNA. If they match correctly, the bubble is enlarged and the remainder of the crRNA binds with its corresponding target DNA, forming what is known as an “R-loop” structure.

Once the R-loop is completely formed, the CRISPR Cascade complex undergoes a conformational change that locks the DNA into place. It also creates a bulge in the second, non-target strand of DNA, which is run through a separate location on the Cascade complex.

Only when a full R-loop state is formed does the Cas3 enzyme bind and cut the DNA at the bulge created in the non-target DNA strand.

The findings reveal an elaborate redundancy to ensure precision and avoid mistakenly chopping up the bacteria’s own DNA.

CRISPR forms a “seed bubble” state, which acts as an initial fail-safe mechanism to ensure that CRISPR RNA matches its target DNA. Image: Liao Lab/HMS

“To apply CRISPR in human medicine, we must be sure the system is accurate and that it does not target the wrong genes,” said Ke, who is co-senior author of the study. “Our argument is that the CRISPR-Cas3 subtype has evolved to be a precise system that carries the potential to be a more accurate system to use for gene editing. If there is mistargeting, we know how to manipulate the system because we know the steps involved and where we might need to intervene.”

Setting the sights

Structures of CRISPR Cascade without target DNA and in its post-R-loop conformational states have been described, but this study is the first to reveal the full sequence of events from seed bubble formation to R-loop formation at high resolution.

In contrast to the scalpel-like Cas9, CRISPR-Cas3 acts like a shredder that chews DNA up beyond repair. While CRISPR-Cas3 has, thus far, limited utility for precision gene editing, it is being developed as a tool to combat antibiotic-resistant strains of bacteria. A better understanding of its mechanisms may broaden the range of potential applications for CRISPR-Cas3.

In addition, all CRISPR-Cas subtypes utilize some version of an R-loop formation to detect and prepare target DNA for cleavage. The improved structural understanding of this process can now enable researchers to work toward modifying multiple types of CRISPR-Cas systems to improve their accuracy and reduce the chance of off-target effects in biomedical applications.

“Scientists hypothesized that these states existed but they were lacking the visual proof of their existence,” said co-first author Min Luo, postdoctoral fellow in the Liao lab at HMS. “The main obstacles came from stable biochemical reconstitution of these states and high-resolution structural visualization. Now, seeing really is believing.”

“We’ve found that these steps must occur in a precise order,” Luo said. “Evolutionarily, this mechanism is very stringent and has triple redundancy, to ensure that this complex degrades only invading DNA.”

Additional authors on the study include Yibei Xiao, Robert P. Hayes, Jonathan Kim, Sherwin Ng, and Fang Ding.

This work is supported by National Institutes of Health

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Established in 1782, Harvard Medical School began with a handful of students and a faculty of three. The first classes were held in Harvard Hall in Cambridge, long before the school’s iconic quadrangle was built in Boston. With each passing decade, the school’s faculty and trainees amassed knowledge and influence, shaping medicine in the United States and beyond. Some community members—and their accomplishments—have assumed the status of legend. We invite you to access the following resources to explore Harvard Medical School’s rich history.

Harvard is the oldest institution of higher education in the United States, established in 1636 by vote of the Great and General Court of the Massachusetts Bay Colony. It was named after the College’s first benefactor, the young minister John Harvard of Charlestown, who upon his death in 1638 left his library and half his estate to the institution. A statue of John Harvard stands today in front of University Hall in Harvard Yard, and is perhaps the University’s best known landmark.

Harvard University has 12 degree-granting Schools in addition to the Radcliffe Institute for Advanced Study. The University has grown from nine students with a single master to an enrollment of more than 20,000 degree candidates including undergraduate, graduate, and professional students. There are more than 360,000 living alumni in the U.S. and over 190 other countries.

From Salk: “Brain’s immune cells linked to Alzheimer’s, Parkinson’s, schizophrenia”

Salk Institute for Biological Studies

Salk and UC San Diego scientists conducted vast microglia survey, revealing links to neurodegenerative diseases and psychiatric illnesses.

Scientists have, for the first time, characterized the molecular markers that make the brain’s front lines of immune defense–cells called microglia–unique. In the process, they discovered further evidence that microglia may play roles in a variety of neurodegenerative and psychiatric illnesses, including Alzheimer’s, Parkinson’s and Huntington’s diseases as well as schizophrenia, autism and depression.

“Microglia are the immune cells of the brain, but how they function in the human brain is not well understood,” says Rusty Gage, professor in Salk’s Laboratory of Genetics, the Vi and John Adler Chair for Research on Age-Related Neurodegenerative Disease, and a senior author of the new work. “Our work not only provides links to diseases but offers a jumping off point to better understand the basic biology of these cells.”

Genes that have previously been linked to neurological diseases are turned on at higher levels in microglia compared to other brain cells, the team reported in Science on May 25, 2017. While the link between microglia and a number of disorders has been explored in the past, the new study offers a molecular basis for this connection.

“These studies represent the first systematic effort to molecularly decode microglia,” says Christopher Glass, a Professor of Cellular and Molecular Medicine and Professor of Medicine at University of California San Diego, also senior author of the paper. “Our findings provide the foundations for understanding the underlying mechanisms that determine beneficial or pathological functions of these cells.”

Microglia are a type of macrophage, white blood cells found throughout the body that can destroy pathogens or other foreign materials. They’re known to be highly responsive to their surroundings and respond to changes in the brain by releasing pro-inflammatory or anti-inflammatory signals. They also prune back the connections between neurons when cells are damaged or diseased. But microglia are notoriously hard to study. They can’t be easily grown in a culture dish and quickly die outside of a living brain.

Nicole Coufal, a pediatric critical care doctor at UC San Diego, who also works in the Gage lab at Salk, wanted to make microglia from stem cells. But she realized there wasn’t any way to identify whether the resulting cells were truly microglia.

“There was not a unique marker that differentiated microglia from circulating macrophages in the rest of the body,” she says.

David Gosselin and Dylan Skola in the Glass lab, together with Coufal and their collaborators, set out to characterize the molecular characteristics of microglia. They worked with neurosurgeons at UC San Diego to collect brain tissue from 19 patients, all of who were having brain surgery for epilepsy, a brain tumor or a stroke. They isolated microglia from areas of tissue that were unaffected by disease, as well as from mouse brains, and then set out to study the cells. The work was made possible by a multidisciplinary collaboration between bench scientists, bioinformaticians and clinicians.

The team used a variety of molecular and biochemical tests–performed within hours of the cells being collected–to characterize which genes are turned on and off in microglia, how the DNA is marked up by regulatory molecules, and how these patterns change when the cells are cultured.

Microglia, they found, have hundreds of genes that are more highly expressed than other types of macrophages, as well as distinct patterns of gene expression compared to other types of brain cells. After the cells were cultured, however, the gene patterns of the microglia began to change. Within just six hours, more than 2,000 genes had their expression turned down by at least fourfold. The results underscore how dependent microglia are on their surroundings in the brain, and why researchers have struggled to culture them.

Next, the researchers analyzed whether any of the genes that were upregulated in microglia compared to other cells had been previously implicated in disease. Genes linked to a variety of neurodegenerative and psychiatric diseases, they found, were highly expressed in microglia.

“A really high proportion of genes linked to multiple sclerosis, Parkinson’s and schizophrenia are much more highly expressed in microglia than the rest of the brain,” says Coufal. “That suggests there’s some kind of link between microglia and the diseases.”

For Alzheimer’s, more than half of the genes known to affect a person’s risk of developing the disease were expressed more highly in microglia than other brain cells.

In mice, however, many of the disease genes weren’t as highly expressed in microglia. “That tells us that maybe mice aren’t the best model organisms for some of these diseases,” Coufal says.

More work is needed to understand exactly how microglia may be altered in people with diseases, but the new molecular profile of microglia offers a way for researchers to begin trying to better culture the cells, or coax stem cells to develop into microglia for future studies.

Other researchers on the study were Baptiste Jaeger, Carolyn O’Connor, Conor Fitzpatrick, Monique Pena, and Amy Adair of the Salk Institute; Inge Holtman, Johannes Schlachetzki, Eniko Sajti, Martina Pasillas, David Gona, and Michael Levy of the University of California San Diego; and Richard Ransohoff of Biogen.

The work and the researchers involved were supported by grants from the Larry L. Hillblom Foundation, National Institutes of Health, Canadian Institute of Health Research, Multiple Sclerosis Society of Canada, University of California San Diego, Dutch MS Research Foundation, the Gemmy and Mibeth Tichelaar Foundation, the DFG, the JPB Foundation, Dolby Family Ventures, The Paul G. Allen Family Foundation, the Engman Foundation, the Ben and Wanda Hildyard Chair in Hereditary Diseases.

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Every cure has a starting point. Like Dr. Jonas Salk when he conquered polio, Salk scientists are dedicated to innovative biological research. Exploring the molecular basis of diseases makes curing them more likely. In an outstanding and unique environment we gather the foremost scientific minds in the world and give them the freedom to work collaboratively and think creatively. For over 50 years this wide-ranging scientific inquiry has yielded life-changing discoveries impacting human health. We are home to Nobel Laureates and members of the National Academy of Sciences who train and mentor the next generation of international scientists. We lead biological research. We prize discovery. Salk is where cures begin.

From NYT: “In ‘Enormous Success,’ Scientists Tie 52 Genes to Human Intelligence”

The New York Times

MAY 22, 2017
Carl Zimmer

Blood samples from some participants in a new study of genes linked to intelligence were held at the U.K. Biobank, above. Credit Wellcome Trust

In a significant advance in the study of mental ability, a team of European and American scientists announced on Monday that they had identified 52 genes linked to intelligence in nearly 80,000 people.

These genes do not determine intelligence, however. Their combined influence is minuscule, the researchers said [Nature Genetics], suggesting that thousands more are likely to be involved and still await discovery. Just as important, intelligence is profoundly shaped by the environment.

Still, the findings could make it possible to begin new experiments into the biological basis of reasoning and problem-solving, experts said. They could even help researchers determine which interventions would be most effective for children struggling to learn.

“This represents an enormous success,” said Paige Harden, a psychologist at the University of Texas, who was not involved in the study.

For over a century, psychologists have studied intelligence by asking people questions. Their exams have evolved into batteries of tests, each probing a different mental ability, such as verbal reasoning or memorization.

In a typical test, the tasks might include imagining an object rotating, picking out a shape to complete a figure, and then pressing a button as fast as possible whenever a particular type of word appears.

Each test-taker may get varying scores for different abilities. But over all, these scores tend to hang together — people who score low on one measure tend to score low on the others, and vice versa. Psychologists sometimes refer to this similarity as general intelligence.

It’s still not clear what in the brain accounts for intelligence. Neuroscientists have compared the brains of people with high and low test scores for clues, and they’ve found a few.

Brain size explains a small part of the variation, for example, although there are plenty of people with small brains who score higher than others with bigger brains.

Other studies hint that intelligence has something to do with how efficiently a brain can send signals from one region to another.

Danielle Posthuma, a geneticist at Vrije University Amsterdam and senior author of the new paper, first became interested in the study of intelligence in the 1990s. “I’ve always been intrigued by how it works,” she said. “Is it a matter of connections in the brain, or neurotransmitters that aren’t sufficient?”

Dr. Posthuma wanted to find the genes that influence intelligence. She started by studying identical twins who share the same DNA. Identical twins tended to have more similar intelligence test scores than fraternal twins, she and her colleagues found.

Hundreds of other studies have come to the same conclusion, showing a clear genetic influence on intelligence [Nature Genetics]. But that doesn’t mean that intelligence is determined by genes alone.

Our environment exerts its own effects, only some of which scientists understand well. Lead in drinking water, for instance, can drag down test scores. In places where food doesn’t contain iodine, giving supplements to children can raise scores.

Advances in DNA sequencing technology raised the possibility that researchers could find individual genes underlying differences in intelligence test scores. Some candidates were identified in small populations, but their effects did not reappear in studies on larger groups.

So scientists turned to what’s now called the genome-wide association study: They sequence bits of genetic material scattered across the DNA of many unrelated people, then look to see whether people who share a particular condition — say, a high intelligence test score — also share the same genetic marker.

In 2014, Dr. Posthuma was part of a large-scale study of over 150,000 people that revealed 108 genes linked to schizophrenia. But she and her colleagues had less luck with intelligence, which has proved a hard nut to crack for a few reasons.

Standard intelligence tests can take a long time to complete, making it hard to gather results on huge numbers of people. Scientists can try combining smaller studies, but they often have to merge different tests together, potentially masking the effects of genes.

As a result, the first generation of genome-wide association studies on intelligence failed to find any genes. Later studies managed to turn up promising results, but when researchers turned to other groups of people, the effect of the genes again disappeared.

But in the past couple of years, larger studies relying on new statistical methods finally have produced compelling evidence that particular genes really are involved in shaping human intelligence.

“There’s a huge amount of real innovation going on,” said Stuart J. Ritchie, a geneticist at the University of Edinburgh who was not involved in the new study.

Dr. Posthuma and other experts decided to merge data from 13 earlier studies, forming a vast database of genetic markers and intelligence test scores. After so many years of frustration, Dr. Posthuma was pessimistic it would work.

“I thought, ‘Of course we’re not going to find anything,’” she said.

She was wrong. To her surprise, 52 genes emerged with firm links to intelligence. A dozen had turned up in earlier studies, but 40 were entirely new.

But all of these genes together account for just a small percentage of the variation in intelligence test scores, the researchers found; each variant raises or lowers I.Q. by only a small fraction of a point.

“It means there’s a long way to go, and there are going to be a lot of other genes that are going to be important,” Dr. Posthuma said.

Christopher F. Chabris, a co-author of the new study at Geisinger Health System in Danville, Pa., was optimistic that many of those missing genes would come to light, thanks to even larger studies involving hundreds of thousands, perhaps millions, of people.

“It’s just like astronomy getting better with bigger telescopes,” he said.

In the new study, Dr. Posthuma and her colleagues limited their research to people of European descent because that raised the odds of finding common genetic variants linked to intelligence.

But other gene studies have shown that variants in one population can fail to predict what people are like in other populations. Different variants turn out to be important in different groups, and this may well be the case with intelligence.

“If you try to predict height using the genes we’ve identified in Europeans in Africans, you’d predict all Africans are five inches shorter than Europeans, which isn’t true,” Dr. Posthuma said.

Studies like the one published today don’t mean that intelligence is fixed by our genes, experts noted. “If we understand the biology of something, that doesn’t mean we’re putting it down to determinism,” Dr. Ritchie said.

As an analogy, he noted that nearsightedness is strongly influenced by genes. But we can change the environment — in the form of eyeglasses — to improve people’s eyesight.

Dr. Harden predicted that an emerging understanding of the genetics of intelligence would make it possible to find better ways to help children develop intellectually. Knowing people’s genetic variations would help scientists measure how effective different strategies are.

Still, Dr. Harden said, we don’t have to wait for such studies to change people’s environments for the better. “We know that lead harms children’s intellectual abilities,” she said. “There’s low-hanging policy fruit here.”

For her part, Dr. Posthuma wants to make sense of the 52 genes she and her colleagues discovered. There are intriguing overlaps between their influence on intelligence and on other traits.

The genetic variants that raise intelligence also tend to pop up more frequently in people who have never smoked. Some of them also are found more often in people who take up smoking but quit successfully.

As for what the genes actually do, Dr. Posthuma can’t say. Four of them are known to control the development of cells, for example, and three do an assortment of things inside neurons.

To understand what makes these genes special, scientists may need to run experiments on brain cells. One possibility would be to take cells from people with variants that predict high and low intelligence.

She and her colleagues might coax them to develop into neurons, which could then grow into “mini-brains” — clusters of neurons that exchange signals in the laboratory. Researchers could then see if their genetic differences made them behave differently.

“We can’t do it overnight,” Dr. Posthuma said, “but it’s something I hope to be able to do in the future.”

See the full article here .

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From EarthSky: “Molecular clocks track human evolution”

EarthSky

April 9, 2017
Bridget Alex, Harvard University
Priya Moorjani, Columbia University

Our cells have a built-in genetic clock, tracking time… but how accurately?. Image via http://www.shutterstock.com

DNA holds the story of our ancestry – how we’re related to the familiar faces at family reunions as well as more ancient affairs: how we’re related to our closest nonhuman relatives, chimpanzees; how Homo sapiens mated with Neanderthals; and how people migrated out of Africa, adapting to new environments and lifestyles along the way. And our DNA also holds clues about the timing of these key events in human evolution. The Conversation

When scientists say that modern humans emerged in Africa about 200,000 years ago and began their global spread about 60,000 years ago, how do they come up with those dates? Traditionally researchers built timelines of human prehistory based on fossils and artifacts, which can be directly dated with methods such as radiocarbon dating and Potassium-argon dating. However, these methods require ancient remains to have certain elements or preservation conditions, and that is not always the case. Moreover, relevant fossils or artifacts have not been discovered for all milestones in human evolution.

Analyzing DNA from present-day and ancient genomes provides a complementary approach for dating evolutionary events. Because certain genetic changes occur at a steady rate per generation, they provide an estimate of the time elapsed. These changes accrue like the ticks on a stopwatch, providing a “molecular clock.” By comparing DNA sequences, geneticists can not only reconstruct relationships between different populations or species but also infer evolutionary history over deep timescales.

Molecular clocks are becoming more sophisticated, thanks to improved DNA sequencing, analytical tools and a better understanding of the biological processes behind genetic changes. By applying these methods to the ever-growing database of DNA from diverse populations (both present-day and ancient), geneticists are helping to build a more refined timeline of human evolution.

How DNA accumulates changes

Molecular clocks are based on two key biological processes that are the source of all heritable variation: mutation and recombination.

Mutations are changes to the DNA code, such as when one nucleotide base (A, T, G or C) is incorrectly subbed for another.. Image via http://www.shutterstock.com

Mutations are changes to the letters of DNA’s genetic code – for instance, a nucleotide Guanine (G) becomes a Thymine (T). These changes will be inherited by future generations if they occur in eggs, sperm or their cellular precursors (the germline). Most result from mistakes when DNA copies itself during cell division, although other types of mutations occur spontaneously or from exposure to hazards like radiation and chemicals.

In a single human genome, there are about 70 nucleotide changes per generation – minuscule in a genome made up of six billion letters. But in aggregate, over many generations, these changes lead to substantial evolutionary variation.

Scientists can use mutations to estimate the timing of branches in our evolutionary tree. First they compare the DNA sequences of two individuals or species, counting the neutral differences that don’t alter one’s chances of survival and reproduction. Then, knowing the rate of these changes, they can calculate the time needed to accumulate that many differences. This tells them how long it’s been since the individuals shared ancestors.

Comparison of DNA between you and your sibling would show relatively few mutational differences because you share ancestors – mom and dad – just one generation ago. However, there are millions of differences between humans and chimpanzees; our last common ancestor lived over six million years ago.

Bits of the chromosomes from your mom and your dad recombine as your DNA prepares to be passed on. Chromosomes image via http://www.shutterstock.com.

Recombination, also known as crossing-over, is the other main way DNA accumulates changes over time. It leads to shuffling of the two copies of the genome (one from each parent), which are bundled into chromosomes. During recombination, the corresponding (homologous) chromosomes line up and exchange segments, so the genome you pass on to your children is a mosaic of your parents’ DNA.

In humans, about 36 recombination events occur per generation, one or two per chromosome. As this happens every generation, segments inherited from a particular individual get broken into smaller and smaller chunks. Based on the size of these chunks and frequency of crossovers, geneticists can estimate how long ago that individual was your ancestor.

Gene flow between divergent populations leads to chromosomes with mosaic ancestry. As recombination occurs in each generation, the bits of Neanderthal ancestry in modern human genomes becomes smaller and smaller over time. Image via Bridget Alex.

Building timelines based on changes

Genetic changes from mutation and recombination provide two distinct clocks, each suited for dating different evolutionary events and timescales.

Because mutations accumulate so slowly, this clock works better for very ancient events, like evolutionary splits between species. The recombination clock, on the other hand, ticks at a rate appropriate for dates within the last 100,000 years. These “recent” events (in evolutionary time) include gene flow between distinct human populations, the rise of beneficial adaptations or the emergence of genetic diseases.

The case of Neanderthals illustrates how the mutation and recombination clocks can be used together to help us untangle complicated ancestral relationships. Geneticists estimate that there are 1.5-2 million mutational differences between Neanderthals and modern humans. Applying the mutation clock to this count suggests the groups initially split between 750,000 and 550,000 years ago.

At that time, a population – the common ancestors of both human groups – separated geographically and genetically. Some individuals of the group migrated to Eurasia and over time evolved into Neanderthals. Those who stayed in Africa became anatomically modern humans.

An evolutionary tree displays the divergence and interbreeding dates that researchers estimated with molecular clock methods for these groups. Image via Bridget Alex.

However, their interactions were not over: Modern humans eventually spread to Eurasia and mated with Neanderthals. Applying the recombination clock to Neanderthal DNA retained in present-day humans, researchers estimate that the groups interbred between 54,000 and 40,000 years ago. When scientists analyzed a Homo sapiens fossil, known as Oase 1, who lived around 40,000 years ago, they found large regions of Neanderthal ancestry embedded in the Oase genome, suggesting that Oase had a Neanderthal ancestor just four to six generations ago. In other words, Oase’s great-great-grandparent was a Neanderthal.

Comparing chromosome 6 from the 40,000-year-old Oase fossil to a present-day human. The blue bands represent segments of Neanderthal DNA from past interbreeding. Oase’s segments are longer because he had a Neanderthal ancestor just 4–6 generations before he lived, based on estimates using the recombination clock. Image via Bridget Alex.

The challenges of unsteady clocks

Molecular clocks are a mainstay of evolutionary calculations, not just for humans but for all forms of living organisms. But there are some complicating factors.

The main challenge arises from the fact that mutation and recombination rates have not remained constant over human evolution. The rates themselves are evolving, so they vary over time and may differ between species and even across human populations, albeit fairly slowly. It’s like trying to measure time with a clock that ticks at different speeds under different conditions.

One issue relates to a gene called Prdm9, which determines the location of those DNA crossover events. Variation in this gene in humans, chimpanzees and mice has been shown to alter recombination hotspots – short regions of high recombination rates. Due to the evolution of Prdm9 and hotspots, the fine-scale recombination rates differ between humans and chimps, and possibly also between Africans and Europeans. This implies that over different timescales and across populations, the recombination clock ticks at slightly different rates as hotspots evolve.

Another issue is that mutation rates vary by sex and age. As fathers get older, they transmit a couple extra mutations to their offspring per year. The sperm of older fathers has undergone more rounds of cell division, so more opportunities for mutations. Mothers, on the other hand, transmit fewer mutations (about 0.25 per year) as a female’s eggs are mostly formed all at the same time, before her own birth. Mutation rates also depend on factors like onset of puberty, age at reproduction and rate of sperm production. These life history traits vary across living primates and probably also differed between extinct species of human ancestors.

Consequently, over the course of human evolution, the average mutation rate seems to have slowed significantly. The average rate over millions of years since the split of humans and chimpanzees has been estimated as about 1×10?? mutations per site per year – or roughly six altered DNA letters per year. This rate is determined by dividing the number of nucleotide differences between humans and other apes by the date of their evolutionary splits, as inferred from fossils. It’s like calculating your driving speed by dividing distance traveled by time passed. But when geneticists directly measure nucleotide differences between living parents and children (using human pedigrees), the mutation rate is half the other estimate: about 0.5×10?? per site per year, or only about three mutations per year.

For the divergence between Neanderthals and modern humans, the slower rate provides an estimate between 765,000-550,000 years ago. The faster rate, however, would suggest half that age, or 380,000-275,000 years ago: a big difference.

To resolve the question of which rates to use when and on whom, researchers have been developing new molecular clock methods, which address the challenges of evolving mutation and recombination rates.

New approaches for better dating

One approach is to focus on mutations that arise at a steady rate regardless of sex, age and species. This may be the case for a special type of mutation that geneticists call CpG transitions by which the C nucelotides spontaneously become T’s. Because CpG transitions mostly do not result from DNA copying errors during cell division, their rates should be mainly independent of life history variables – and presumably more uniform over time.

Focusing on CpG transitions, geneticists recently estimated the split between humans and chimps to have occurred between 9.3 and 6.5 million years ago, which agrees with the age expected from fossils. While in comparisons across species, these mutations seem to happen more like clockwork than other types, they are still not completely steady.

Another approach is to develop models that adjust molecular clock rates based on sex and other life history traits. Using this method, researchers calculated a chimp-human divergence consistent with the CpG estimate and fossil dates. The drawback here is that, when it comes to ancestral species, we can’t be sure of life history traits, like age at puberty or generation length, leading to some uncertainty in the estimates.

The most direct solution comes from analyses of ancient DNA recovered from fossils. Because the fossil specimens are independently dated by geologic methods, geneticists can use them to calibrate the molecular clocks for a given time period or population.

This strategy recently resolved the debate over the timing of our divergence with Neanderthals. In 2016, geneticists extracted ancient DNA from 430,000-year-old fossils that were Neanderthal ancestors, after their lineage split from Homo sapiens. Knowing where these fossils belong in the evolutionary tree, geneticists could confirm that for this period of human evolution, the slower molecular clock rate of 0.5×10?? provides accurate dates. That puts the Neanderthal-modern human split between 765,000 to 550,000 years ago.

As geneticists sort out the intricacies of molecular clocks and sequence more genomes, we’re poised to learn more than ever about human evolution, directly from our DNA.

Bridget Alex, Postdoctoral College Fellow, Department of Human Evolutionary Biology, Harvard University and Priya Moorjani, Postdoctoral Research Fellow in Biological Sciences, Columbia University

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From Nautilus: “The Man Who Kicked Off the Biotech Revolution” Hamilton Smith

Nautilus

3.5.17
Carl Zimmer

It’s hard to tell precisely how big a role biotechnology plays in our economy, because it infiltrates so many parts of it. Genetically modified organisms such as microbes and plants now create medicine, food, fuel, and even fabrics. Recently, Robert Carlson, of the biotech firm Biodesic and the investment firm Bioeconomy Capital, decided to run the numbers and ended up with an eye-popping estimate. He concluded that in 2012, the last year for which good data are available, revenues from biotechnology in the United States alone were over $324 billion.

“If we talk about mining or several manufacturing sectors, biotech is bigger than those,” said Carlson. “I don’t think people appreciate that.”

Matchmaker Biotech pioneer Hamilton Smith chose to study recombination in a species of bacteria called Haemophilus influenza (above), which can take up foreign DNA fragments and integrate them into its own DNA. Media for Medical/UIG via Getty Images

What makes the scope of biotech so staggering is not just its size, but its youth. Manufacturing first exploded in the Industrial Revolution of the 19th century. But biotech is only about 40 years old. It burst into existence thanks largely to a discovery made in the late 1960s by Hamilton Smith, a microbiologist then at Johns Hopkins University, and his colleagues, that a protein called a restriction enzyme can slice DNA. Once Smith showed the world how restriction enzymes work, other scientists began using them as tools to alter genes.

“And once you have the ability to start to manipulate the world with those tools,” said Carlson, “the world opens up.”

The story of restriction enzymes is a textbook example of how basic research can ultimately have a huge impact on society. Smith had no such grand ambitions when he started his work. He just wanted to do some science. “I was just having a lot of fun, learning as I went,” Smith, now 85, said.

In 1968, when Smith was a new assistant professor at Johns Hopkins University, he became curious about how cells cut DNA into pieces and shuffle them into new arrangements—a process known as recombination. “It’s a universal thing,” Smith said. “Every living thing has recombination systems. But at the time, no one was sure how it worked, mechanically.”

Smith chose to study recombination in a species of bacteria called Haemophilus influenza. Like many other species, H. influenzae can take up foreign DNA, either sucking in loose fragments from the environment or gaining them from microbial donors. Somehow, the bacterium can then integrate these fragments into its own DNA.

Bacteria gain useful genes in this way, endowing them with new traits such as resistance to antibiotics. But recombination also has a dark side for H. influenzae. Invading viruses can hijack the recombination machinery in bacteria. They then insert their own genes into their host’s DNA, so that the microbes make new copies of the virus.

To understand recombination, Smith produced radioactive viruses by introducing viruses into bacteria that had been fed radioactive phosphorus. New viruses produced inside the bacteria ended up with radioactive phosphorus in their DNA. Smith and his colleagues could then unleash these radioactive viruses on other bacteria. The scientists expected that during the infection, the bacteria’s genes would become radioactive as the viruses inserted their genetic material into their host’s DNA.

At least that was they thought would happen. When Smith’s graduate student Kent Wilcox infected bacteria with the radioactive viruses, the radioactivity never ended up in the bacteria’s own genome.

Trying to make sense of the failure, Wilcox suggested to Smith that the bacteria were destroying the viral DNA. He based his suggestion on a hypothesis proposed a few years earlier by Werner Arber, a microbiologist at the University of Geneva. Arber speculated that enzymes could restrict the growth of viruses by chopping up their DNA, and dubbed these hypothetical molecules “restriction enzymes.”

Arber recognized that if restriction enzymes went on an unchecked rampage, they could kill the bacteria themselves by chopping up their own DNA. He speculated that bacteria were shielding their own DNA from assault, and thus avoiding suicide, by covering their genes with carbon and hydrogen atoms—a process known as methylation. The restriction enzymes couldn’t attack methylated DNA, Arber proposed, but it could attack the unprotected DNA of invading viruses.

The week before Wilcox had carried out his baffling experiment, Smith had assigned his lab a provocative new paper supporting Arber’s hypothesis. Matthew Meselson and Robert Yuan at Harvard University reported in the paper how they had discovered a protein in E. coli that cut up foreign DNA—in other words, an actual restriction enzyme. With that paper fresh in his mind, Wilcox suggested to Smith that they had just stumbled across another restriction enzyme in Haemophilus influenzae.

Smith tested the idea with an elegant experiment. He poured viral DNA into a test tube, and DNA from H. influenza into another. To each of these tubes, he then added a soup of proteins from the bacteria. If indeed the bacteria made restriction enzymes, the enzymes in the soup would chop up the viral DNA into small pieces.

Scientists were decades away from inventing the powerful sequencers that are used today to analyze DNA. But Smith came up with a simple way to investigate the DNA in his test tubes. A solution containing large pieces of DNA is more viscous—more syrupy, in effect—than one with small pieces. So Smith measured the solution in his two test tubes with a device called a viscometer. As he had predicted, the virus DNA quickly became far less viscous. Something—some H. influenzae protein, presumably—was cutting the virus DNA into little pieces.

“So I immediately knew this had to be a restriction enzyme,” Smith said. “It was a wonderful result—five minutes, and you know you have a discovery.”

That instant gratification was followed by months of tedium, as Smith and his colleagues sorted through the proteins in their cell extracts until at last they identified a restriction enzyme. They also discovered a methylation enzyme that protected H. influenzae’s own DNA from destruction by shielding it with carbon and hydrogen.

Once Smith and his colleagues published the remarkable details of their restriction enzymes, other scientists began to investigate them as well. They didn’t just study the enzymes, though—they began employing them as a tool. In 1972, Paul Berg, a biologist at Stanford University, used restriction enzymes to make cuts in the DNA of SV40 viruses, and then used other enzymes to attach the DNA from another virus to those loose ends. Berg thus created a single piece of DNA made up of genetic material from two species.

A pack of scientists followed Berg’s lead. They realized that they could use restriction enzymes to insert genes from many different species into bacteria, which could then churn out proteins from those genes. In effect, bacteria could be transformed into biological factories.

In 1978, Hamilton Smith got a call from Stockholm. He learned that he was sharing that year’s Nobel Prize in Medicine with Werner Arber and Daniel Nathans, another Johns Hopkins scientist who had followed up on Smith’s enzyme research with experiments of his own. Smith was as flummoxed as he was delighted.

“They caught me off-guard,” Smith said. “I always looked up to the Nobelists as being incredibly smart people who had accomplished some world-shaking thing. It just didn’t seem like I was in that league.”

But already the full impact of his work was starting to become clear. Companies sprouted up that were dedicated to using restriction enzymes to modify DNA. The first commercial application of this technology came from Genentech, a company founded in 1976. Genentech scientists used restriction enzymes to create a strain of E. coli that carried the gene for human insulin. Previously, people with diabetes could only purchase insulin extracted from the pancreases of cows and pigs. Genentech sold insulin produced by swarms of bacteria reared in giant metal drums.

Over the years, scientists have built on Smith’s initial successes by finding new tools for manipulating DNA. Yet even today, researchers make regular use of restriction enzymes to slice open genes. “They’re still absolutely crucial,” said Carlson. “If you want to put a specific sequence of DNA in another sequence, it’s still most often restriction enzymes that you use to do that.”

And as Smith has watched restriction enzymes become powerful and versatile, he has slowly overcome his case of Nobel imposter syndrome. “It probably was okay to get it,” he admitted.

See the full article here .

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From NYT: “A Family’s Shared Defect Sheds Light on the Human Genome”

The New York Times

JAN. 9, 2017
NATALIE ANGIER

Headcase Design

They said it was their family curse: a rare congenital deformity called syndactyly, in which the thumb and index finger are fused together on one or both hands. Ten members of the extended clan were affected, and with each new birth, they told Stefan Mundlos of the Max Planck Institute for Molecular Genetics, the first question was always: “How are the baby’s hands? Are they normal?”

Afflicted relatives described feeling like outcasts in their village, convinced that their “strange fingers” repulsed everybody they knew — including their unaffected kin. “One woman told me that she never received a hug from her father,” Dr. Mundlos said. “He avoided her.”

The family, under promise of anonymity, is taking part in a study by Dr. Mundlos and his colleagues of the origin and development of limb malformations. And while the researchers cannot yet offer a way to prevent syndactyly, or to entirely correct it through surgery, Dr. Mundlos has sought to replace the notion of a family curse with “a rational answer for their condition,” he said — and maybe a touch of pioneers’ pride.

The scientists have traced the family’s limb anomaly to a novel class of genetic defects unlike any seen before, a finding with profound implications for understanding a raft of heretofore mysterious diseases.

The mutations affect a newly discovered design feature of the DNA molecule called topologically associating domains, or TADs. It turns out that the vast informational expanse of the genome is divvied up into a series of manageable, parochial and law-abiding neighborhoods with strict nucleic partitions between them — each one a TAD.

The hand of a woman with syndactyly, the congenital fusion of fingers. The deformity may range from a slight degree of webbing to almost complete fusion as shown here. Credit SPL/Science Source

Breach a TAD barrier, and you end up with the molecular equivalent of that famous final scene in Mel Brooks’s comedy, “Blazing Saddles,” when the cowboy actors from one movie set burst through a wall and onto the rehearsal stage of a campy Fred Astaire-style musical. Soon fists, top hats and cream pies are flying.

By studying TADs, researchers hope to better fathom the deep structure of the human genome, in real time and three dimensions, and to determine how a quivering, mucilaginous string of some three-billion chemical subunits that would measure more than six-feet long if stretched out nonetheless can be coiled and compressed down to four-10,000ths of an inch, the width of a cell nucleus — and still keep its operational wits about it.

“DNA is a super-long molecule packed into a very small space, and it’s clear that it’s not packed randomly,” Dr. Mundlos said. “It follows a very intricate and controlled packing mechanism, and TADs are a major part of the folding protocol.”

For much of the past 50 years, genetic research has focused on DNA as a kind of computer code, a sequence of genetic “letters” that inscribe instructions for piecing together amino acids into proteins, which in turn do the work of keeping us alive.

Read Between the Folds

Most of the genetic diseases deciphered to date have been linked to mishaps in one or another protein recipe. Scanning the DNA of patients with Duchenne muscular dystrophy, for example, scientists have identified telltale glitches in the gene that encodes dystrophin, a protein critical to muscle stability.

At the root of Huntington’s disease, which killed the folk singer Woody Guthrie, are short, repeated bits of nucleic nonsense sullying the code for huntingtin, an important brain protein. The mutant product that results soon shatters into neurotoxic shards.

Yet researchers soon realized there was much more to the genome than the protein codes it enfolded. “We were caught up in the idea of genetic information being linear and one-dimensional,” said Job Dekker, a biologist at the University of Massachusetts Medical School.

For one thing, as the sequencing of the complete human genome revealed, the portions devoted to specifying the components of hemoglobin, collagen, pepsin and other proteins account for just a tiny fraction of the whole, maybe 3 percent of human DNA’s three billion chemical bases.

And there was the restless physicality of the genome, the way it arranged itself during cell division into 23 spindly pairs of chromosomes that could be stained and studied under a microscope, and then somehow, when cell replication was through, merged back together into a baffling, ever-wriggling ball of chromatin — DNA wrapped in a protective packaging of histone proteins.

Stefan Mundlos of the Max Planck Institute for Molecular Genetics in Germany studies the origin and development of limb malformations, some of which are caused by a novel class of genetic defects. Credit Norbert Michalke/Max Planck Institute for Molecular Genetics, Berlin

What was the link, scientists wondered, between the shape and animation of the DNA molecule at any given moment, in any given cell — and every cell has its own copy of the genome — and the relative mouthiness or muteness of the genetic information the DNA holds?

“We realized that in order to understand how genetic information is controlled, we had to figure out how DNA was folded in space,” said Bing Ren of the University of California, San Diego.

Using a breakthrough technology developed by Dr. Dekker and his colleagues called chromosome conformation capture, researchers lately have made progress in tracking the deep structure of DNA. In this approach, chromatin is chemically “frozen” in place, enzymatically chopped up and labeled, and then allowed to reassemble.

The pieces that find each other again, scientists have determined, are those that were physically contiguous in the first place — only now all their positions and relationships are clearly marked.

Through chromosome conformation studies and related research, scientists have discovered the genome is organized into about 2,000 jurisdictions, and they are beginning to understand how these TADs operate.

As with city neighborhoods, TADs come in a range of sizes, from tiny walkable zones a few dozen DNA subunits long to TADs that sprawl over tens of thousands of bases and you’re better off taking the subway. TAD borders serve as folding instructions for DNA. “They’re like the dotted lines on a paper model kit,” Dr. Dekker said.

TAD boundaries also dictate the rules of genetic engagement.

Scientists have long known that protein codes are controlled by an assortment of genetic switches and enhancers — noncoding sequences designed to flick protein production on, pump it into high gear and muzzle it back down again. The new research indicates that switches and enhancers act only on those genes, those protein codes, stationed within their own precincts.

Because TADs can be quite large, the way the Upper West Side of Manhattan comprises an area of about 250 square blocks, a genetic enhancer located at the equivalent of, say, Lincoln Center on West 65th Street, can amplify the activity of a gene positioned at the Cathedral of St. John the Divine, 45 blocks north.

But under normal circumstances, one thing an Upper West Side enhancer will not do is reach across town to twiddle genes residing on the Upper East Side.

Scientists have learned that disruptions of the genome’s boundaries may cause syndactyly and other diseases, including some pediatric brain disorders that affect the brain’s white matter. Credit Living Art Enterprises, LLC/Science Source

“Genes and regulatory elements are like people,” Dr. Dekker said. “They care about and communicate with those in their own domain, and they ignore everything else.”

Breaking Boundaries

What exactly do these boundaries consist of, that manage to both direct the proper folding of the DNA molecule and prevent cross talk between genes and gene switches in different domains? Scientists are not entirely sure, but preliminary results indicate that the boundaries are DNA sequences that attract the attention of sticky, roughly circular proteins called cohesin and CTCF, which adhere thickly to the boundary sequences like insulating tape.

Between those boundary points, those clusters of insulating proteins, the chromatin strand can loop up and over like the ribbon in a birthday bow, allowing genetic elements distributed along the ribbon to touch and interact with one another. But the insulating proteins constrain the movement of each chromatin ribbon, said Richard A. Young of the Whitehead Institute for Biomedical Research, and keep it from getting entangled with neighboring loops — and the genes and regulatory elements located thereon.

The best evidence for the importance of TADs is to see what happens when they break down. Researchers have lately linked a number of disorders to a loss of boundaries between genomic domains, including cancers of the colon, esophagus, brain and blood.

In such cases, scientists have failed to find mutations in any of the protein-coding sequences commonly associated with the malignancies, but instead identified DNA damage that appeared to shuffle around or eliminate TAD boundaries. As a result, enhancers from neighboring estates suddenly had access to genes they were not meant to activate.

Reporting in the journal Science, Dr. Young and his colleagues described a case of leukemia in which a binding site for insulator proteins had been altered not far from a gene called TAL1, which if improperly activated is known to cause leukemia. In this instance, disruption of the nearby binding site, Dr. Young said, “broke up the neighborhood and allowed an outside enhancer to push TAL1 to the point of tumorigenesis,” the production of tumors.

Now that researchers know what to look for, he said, TAD disruptions may prove to be a common cause of cancer. The same may be true of developmental disorders — like syndactyly.

In journals like Cell and Nature, Dr. Mundlos and his co-workers described their studies of congenital limb malformations in both humans and mice. The researchers have detected major TAD boundary disruptions that allowed the wrong control elements to stimulate muscle genes at the wrong time and in the wrong tissue.

“If a muscle gene turns on in the cartilage of developing digits,” Dr. Mundlos said, “you get malformations.”

Edith Heard, director of the genetics and developmental biology department at the Institut Curie in France, who with Dr. Dekker coined the term TAD, said that while researchers were just beginning to get a handle on the architecture of DNA, “suddenly a lot of things are falling into place. We’re coming into a renaissance time for understanding how the genome works.”

See the full article here .

Please help promote STEM in your local schools.

Stem Education Coalition