Research Area: Genome Engineering

​CRISPR, SHERLOCK, REPAIR, RNA editing, DNA editing, brain disorders, diagnostics, disease models, therapeutics

Toward a better understanding of the brain

Anne Trafton |

In 2011, about a month after joining the MIT faculty, Feng Zhang attended a talk by Harvard Medical School Professor Michael Gilmore, who studies the pathogenic bacterium Enteroccocus. The scientist mentioned that these bacteria protect themselves from viruses with DNA-cutting enzymes known as nucleases, which are part of a defense system known as CRISPR.

“I had no idea what CRISPR was but I was interested in nucleases,” Zhang says. “I went to look up CRISPR, and that’s when I realized you might be able to engineer it for use for genome editing.”

Zhang devoted himself to adapting the system to edit genes in mammalian cells and recruited new members to his nascent lab at the Broad Institute of MIT and Harvard to work with him on this project. In January 2013, they reported their success in the journal Science.

Since then, scientists in fields from medicine to plant biology have begun using CRISPR to study gene function and investigate the possibility of correcting faulty genes that cause disease. Zhang now heads a lab of 19 scientists who continue to develop the system and pursue applications of genome editing, especially in neuroscience.

“The goal is to try to make our lives better by developing new technologies and using them to understand biological systems so that we can improve our treatment of disease and our quality of life,” says Zhang, who is also a member of MIT’s McGovern Institute for Brain Research and recently earned tenure in MIT’s Departments of Biological Engineering and Brain and Cognitive Sciences.

Understanding the brain

Growing up in Des Moines, Iowa, where his parents moved from China when he was 11, Zhang had plenty of opportunities to feed his interest in science. He participated in Science Bowl competitions and took special Saturday science classes, where he got his first introduction to molecular biology. Experiments such as extracting DNA from strawberries and transforming bacteria with genes for drug resistance whetted his appetite for genetic engineering, which was further stimulated by a showing of “Jurassic Park.”

“That really caught my attention,” he recalls. “It didn’t seem that far-fetched. I guess that’s what makes it good science fiction. It kind of tantalizes your imagination.”

As a sophomore in high school, Zhang began working with Dr. John Levy in a gene therapy lab at the Iowa Methodist Medical Center in Des Moines, where he studied green fluorescent protein (GFP). Scientists had recently figured out how to adapt this naturally occurring protein to tag and image proteins inside living cells. Zhang used it to track viral proteins within infected cells to determine how the proteins assemble to form new viruses. He also worked on a project to adapt GFP for a different purpose — protecting DNA from damage induced by ultraviolet light.

At Harvard University, where he earned his undergraduate degree, Zhang majored in chemistry and physics and did research under the mentorship of Xiaowei Zhuang, a professor of chemistry and chemical biology. “I was always interested in biology but I felt that it’s important to get a solid training in chemistry and physics,” he says.

While Zhang was at Harvard, a close friend was severely affected by a psychiatric disorder. That experience made Zhang think about whether such disorders could be approached just like cancer or heart disease, if only scientists knew more about their underlying causes.

“The difference is we’re at a much earlier stage of understanding psychiatric diseases. That got me really interested in trying to understand more about how the brain works,” he says.

At Stanford University, where Zhang earned his PhD in chemistry, he worked with Karl Deisseroth, who was just starting his lab with a focus on developing new technology for studying the brain. Zhang was the second student to join the lab, and he began working on a protein called channelrhodopsin, which he and Deisseroth believed held potential for engineering mammalian cells to respond to light.

The resulting technique, known as optogenetics, has transformed biological research. Collaborating with Edward Boyden, a member of the Deisseroth lab who is now a professor at MIT, Zhang adapted channelrhodopsin so that it could be inserted into neurons and make them light-sensitive. Using this approach, neuroscientists can now selectively activate and de-activate specific neurons in the brain, allowing them to map brain circuits and investigate how disruption of those circuits causes disease.

Better gene editing

After leaving Stanford, Zhang spent a year as a junior fellow at the Harvard Society of Fellows, studying brain development with Professor Paola Arlotta and collaborating with Professor George Church. That’s when he began to focus on gene editing — a type of genetic engineering that allows researchers to selectively delete a gene or replace it with a new one.

He began with zinc finger nucleases — enzymes that can be designed to target and cut specific DNA sequences. However, these proteins turned out to be challenging to work with, in part because it is so time-consuming to design a new protein for each possible DNA target.

That led Zhang to experiment with a different type of nucleases known as transcription activator-like effector nucleases (TALENs), but these also proved laborious to work with. “Learning how to use them is a project on its own,” Zhang says.

When he heard about CRISPR in early 2011, Zhang sensed that harnessing the natural bacterial process held the potential to solve many of the challenges associated with those earlier gene-editing techniques. CRISPR includes a nuclease called Cas9, which can be guided to the correct genetic target by RNA molecules known as guide strands. For each target, scientists need only design and synthesize a new RNA guide, which is much simpler than creating new TALEN and zinc finger proteins.

Since his first CRISPR paper in 2013, Zhang’s lab has devised many enhancements to the original system, such as making the targeting more precise and preventing unintended cuts in the wrong locations. They also recently reported another type of CRISPR system based on a different nuclease called Cpf1, which is simpler and has unique features that further expand the genome editing toolbox.

Zhang’s lab has become a hub for CRISPR research worldwide. It has shared CRISPR-Cas9 components in response to nearly 30,000 requests from academic laboratories around the world and has trained thousands of researchers in the use of CRISPR-Cas9 genome-editing technology through in-person events and online opportunities.

His team is now working on creating animal models of autism, Alzheimer’s, and other neurological disorders, and in the long term, they hope to develop CRISPR for use in humans to potentially cure diseases caused by defective genes.

“There are many genetic diseases that we don’t have any way of treating and this could be one way, but we still have to do a lot of work,” Zhang says.

McGovern neuroscientists reverse autism symptoms

Anne Trafton |

Autism has diverse genetic causes, most of which are still unknown. About 1 percent of people with autism are missing a gene called Shank3, which is critical for brain development. Without this gene, individuals develop typical autism symptoms including repetitive behavior and avoidance of social interactions.

In a study of mice, MIT researchers have now shown that they can reverse some of those behavioral symptoms by turning the gene back on later in life, allowing the brain to properly rewire itself.

“This suggests that even in the adult brain we have profound plasticity to some degree,” says Guoping Feng, an MIT professor of brain and cognitive sciences. “There is more and more evidence showing that some of the defects are indeed reversible, giving hope that we can develop treatment for autistic patients in the future.”

Feng, who is the James W. and Patricia Poitras Professor of Neuroscience and a member of MIT’s McGovern Institute for Brain Research and the Stanley Center for Psychiatric Research at the Broad Institute, is the senior author of the study, which appears in the Feb. 17 issue of Nature. The paper’s lead authors are former MIT graduate student Yuan Mei and former Broad Institute visiting graduate student Patricia Monteiro, now at the University of Coimbra in Portugal.

Boosting communication

The Shank3 protein is found in synapses — the connections that allow neurons to communicate with each other. As a scaffold protein, Shank3 helps to organize the hundreds of other proteins that are necessary to coordinate a neuron’s response to incoming signals.

Studying rare cases of defective Shank3 can help scientists gain insight into the neurobiological mechanisms of autism. Missing or defective Shank3 leads to synaptic disruptions that can produce autism-like symptoms in mice, including compulsive behavior, avoidance of social interaction, and anxiety, Feng has previously found. He has also shown that some synapses in these mice, especially in a part of the brain called the striatum, have a greatly reduced density of dendritic spines — small buds on neurons’ surfaces that help with the transmission of synaptic signals.

In the new study, Feng and colleagues genetically engineered mice so that their Shank3 gene was turned off during embryonic development but could be turned back on by adding tamoxifen to the mice’s diet.
When the researchers turned on Shank3 in young adult mice (two to four and a half months after birth), they were able to eliminate the mice’s repetitive behavior and their tendency to avoid social interaction. At the cellular level, the team found that the density of dendritic spines dramatically increased in the striatum of treated mice, demonstrating the structural plasticity in the adult brain.

However, the mice’s anxiety and some motor coordination symptoms did not disappear. Feng suspects that these behaviors probably rely on circuits that were irreversibly formed during early development.
When the researchers turned on Shank3 earlier in life, only 20 days after birth, the mice’s anxiety and motor coordination did improve. The researchers are now working on defining the critical periods for the formation of these circuits, which could help them determine the best time to try to intervene.

“Some circuits are more plastic than others,” Feng says. “Once we understand which circuits control each behavior and understand what exactly changed at the structural level, we can study what leads to these permanent defects, and how we can prevent them from happening.”

Gordon Fishell, a professor of neuroscience at New York University School of Medicine, praises the study’s “elegant approach” and says it represents a major advance in understanding the circuitry and cellular physiology that underlie autism. “The combination of behavior, circuits, physiology, and genetics is state-of-the art,” says Fishell, who was not involved in the research. “Moreover, Dr. Feng’s demonstration that restoration of Shank3 function reverses autism symptoms in adult mice suggests that gene therapy may ultimately prove an effective therapy for this disease.”

Early intervention

For the small population of people with Shank3 mutations, the findings suggest that new genome-editing techniques could in theory be used to repair the defective Shank3 gene and improve these individuals’ symptoms, even later in life. These techniques are not yet ready for use in humans, however.

Feng believes that scientists may also be able to develop more general approaches that would apply to a larger population. For example, if the researchers can identify defective circuits that are specific for certain behavioral abnormalities in some autism patients, and figure out how to modulate those circuits’ activity, that could also help other people who may have defects in the same circuits even though the problem arose from a different genetic mutation.

“That’s why it’s important in the future to identify what subtype of neurons are defective and what genes are expressed in these neurons, so we can use them as a target without affecting the whole brain,” Feng says.

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

Anne Trafton |

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 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, DC. “Many of the safety concerns are related to off-target effects,” he said. “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.”

Feng Zhang describes new system for genome engineering

Anne Trafton |

A team including the scientist who first harnessed the CRISPR-Cas9 system for mammalian genome editing has now identified a different CRISPR system with the potential for even simpler and more precise genome engineering.

In a study published today in Cell, Feng Zhang and his colleagues at the Broad Institute of MIT and Harvard and the McGovern Institute for Brain Research at MIT, with co-authors Eugene Koonin at the National Institutes of Health, Aviv Regev of the Broad Institute and the MIT Department of Biology, and John van der Oost at Wageningen University, describe the unexpected biological features of this new system and demonstrate that it can be engineered to edit the genomes of human cells.

“This has dramatic potential to advance genetic engineering,” says Eric Lander, director of the Broad Institute. “The paper not only reveals the function of a previously uncharacterized CRISPR system, but also shows that Cpf1 can be harnessed for human genome editing and has remarkable and powerful features. The Cpf1 system represents a new generation of genome editing technology.”

CRISPR sequences were first described in 1987, and their natural biological function was initially described in 2010 and 2011. The application of the CRISPR-Cas9 system for mammalian genome editing was first reported in 2013, by Zhang and separately by George Church at Harvard University.

In the new study, Zhang and his collaborators searched through hundreds of CRISPR systems in different types of bacteria, searching for enzymes with useful properties that could be engineered for use in human cells. Two promising candidates were the Cpf1 enzymes from bacterial species Acidaminococcus and Lachnospiraceae, which Zhang and his colleagues then showed can target genomic loci in human cells.

“We were thrilled to discover completely different CRISPR enzymes that can be harnessed for advancing research and human health,” says Zhang, the W.M. Keck Assistant Professor in Biomedical Engineering in MIT’s Department of Brain and Cognitive Sciences.

The newly described Cpf1 system differs in several important ways from the previously described Cas9, with significant implications for research and therapeutics, as well as for business and intellectual property:

  • First: In its natural form, the DNA-cutting enzyme Cas9 forms a complex with two small RNAs, both of which are required for the cutting activity. The Cpf1 system is simpler in that it requires only a single RNA. The Cpf1 enzyme is also smaller than the standard SpCas9, making it easier to deliver into cells and tissues.
  • Second, and perhaps most significantly: Cpf1 cuts DNA in a different manner than Cas9. When the Cas9 complex cuts DNA, it cuts both strands at the same place, leaving “blunt ends” that often undergo mutations as they are rejoined. With the Cpf1 complex the cuts in the two strands are offset, leaving short overhangs on the exposed ends. This is expected to help with precise insertion, allowing researchers to integrate a piece of DNA more efficiently and accurately.
  • Third: Cpf1 cuts far away from the recognition site, meaning that even if the targeted gene becomes mutated at the cut site, it can likely still be recut, allowing multiple opportunities for correct editing to occur.
  • Fourth: The Cpf1 system provides new flexibility in choosing target sites. Like Cas9, the Cpf1 complex must first attach to a short sequence known as a PAM, and targets must be chosen that are adjacent to naturally occurring PAM sequences. The Cpf1 complex recognizes very different PAM sequences from those of Cas9. This could be an advantage in targeting some genomes, such as in the malaria parasite as well as in humans.

“The unexpected properties of Cpf1 and more precise editing open the door to all sorts of applications, including in cancer research,” says Levi Garraway, an institute member of the Broad Institute, and the inaugural director of the Joint Center for Cancer Precision Medicine at the Dana-Farber Cancer Institute, Brigham and Women’s Hospital, and the Broad Institute. Garraway was not involved in the research.

An open approach to empower research

Zhang, along with the Broad Institute and MIT, plan to share the Cpf1 system widely. As with earlier Cas9 tools, these groups will make this technology freely available for academic research via the Zhang lab’s page on the plasmid-sharing website Addgene, through which the Zhang lab has already shared Cas9 reagents more than 23,000 times with researchers worldwide to accelerate research. The Zhang lab also offers free online tools and resources for researchers through its website.

The Broad Institute and MIT plan to offer nonexclusive licenses to enable commercial tool and service providers to add this enzyme to their CRISPR pipeline and services, further ensuring availability of this new enzyme to empower research. These groups plan to offer licenses that best support rapid and safe development for appropriate and important therapeutic uses.

“We are committed to making the CRISPR-Cpf1 technology widely accessible,” Zhang says. “Our goal is to develop tools that can accelerate research and eventually lead to new therapeutic applications. We see much more to come, even beyond Cpf1 and Cas9, with other enzymes that may be repurposed for further genome editing advances.”

 

Broad Institute-MIT team identifies highly efficient new Cas9 for in vivo genome editing

by Broad Institute |

A collaborative study between researchers from the Broad Institute of MIT and Harvard, Massachusetts Institute of Technology, and the National Center for Biotechnology Information of the National Institutes of Health (NIH-NCBI) has identified a highly efficient Cas9 nuclease that overcomes one of the primary challenges to in vivo genome editing. This finding, published today in Nature, is expected to help make the CRISPR toolbox accessible for in vivo experimental and therapeutic applications.

Originally discovered in bacteria, the CRISPR-Cas9 system enables the cutting of DNA as a defense mechanism against viral infection. Although numerous microbial species possess this system, the Cas9 enzyme from Streptococcus pyogenes (SpCas9) was the first to be engineered for altering the DNA of higher organisms, and has since emerged as the basis for a series of highly versatile genome modification technologies.

Smaller packaging

In order to perturb genes in adult animals, key components of the CRISPR-Cas9 system must be introduced into cells using delivery vehicles known as vectors. Adeno-associated virus (AAV) is considered one of the most promising candidate vectors, as it is not known to cause human disease and has already gained clinical regulatory approval in Europe. However, the small cargo capacity of AAV makes it challenging to package both the SpCas9 enzyme and the other components required for gene editing into a single viral particle.

The Cas9 nuclease from the bacteria Staphylococcus aureus (SaCas9), presented in this new work, is 25% smaller than SpCas9, offering a solution to the AAV packaging problem.

The Broad/MIT team, led by Feng Zhang, core member of the Broad Institute and investigator at the McGovern Institute for Brain Research at MIT, along with collaborators at MIT, led by MIT Institute Professor Phillip Sharp, and the NCBI led by Eugene Koonin, set out to identify smaller Cas9 enzymes that could replicate the efficiency of the current SpCas9 system, while allowing packaging into delivery vehicles such as AAV. The researchers began by using comparative genomics to analyze Cas9s from more than 600 different types of bacteria, selecting six smaller enzymes for further study.

“Sifting through the 600 or so available Cas9 sequences, we identified a group of small variants in which the enzymatic domains were intact whereas the non-enzymatic portion was substantially truncated,” said Eugene Koonin, senior investigator with the NCBI and a contributing author of the study. “Luckily, one of these smaller Cas9 proteins turned out to be suitable for the development of the methodology described in this paper. We are now actively exploring the diversity of Cas9 proteins and their relatives in the hope to find new varieties that could potentially lead to even more powerful tools.”

After rigorous testing, only the Cas9 from S. aureus demonstrated DNA cutting efficiency comparable to that of SpCas9 in mammalian cells. The team then used a method known as BLESS, previously developed by Nicola Crosetto of the Karolinska Institute and Ivan Dikic at the Goethe University Medical School, to determine the presence of unintended “off-targets” across the entire genomic space. Again, SaCas9 and SpCas9 demonstrated comparable DNA targeting accuracy.

The team demonstrated the power of in vivo gene editing with AAV/SaCas9-mediated targeting of PCSK9, a promising drug target. The loss of PCSK9 in humans has been associated with the reduced risk of cardiovascular disease and lower levels of LDL cholesterol. In a mouse model, the team observed almost complete depletion of PCKS9 in the blood one week after administration of AAV/SaCas9 and a 40% decrease in total cholesterol. The mice showed no overt signs of inflammation or immune response.

“While we have chosen a therapeutically relevant target, PCSK9, in this proof-of-principle study, the greater goal here is the development of a versatile and efficient system that expands our ability to edit genomes in vivo,” said Fei Ann Ran, co-first author of the study, along with Le Cong and Winston Yan.

More broadly, SaCas9 is expected improve scientists’ ability to screen for the effects of mutations and better understand gene function using animal models. In the future, it may also be engineered to allow the targeted control of gene expression, which can be employed to expand our understanding of transcriptional and epigenetic regulation in the cell.

The next step, says senior author Feng Zhang, is to compare and contrast the two Cas9s in the hope of recognizing ways to further optimize the system.

“This study highlights the power of using comparative genome analysis to expand the CRISPR-Cas9 toolbox,” said Zhang. “Our long-term goal is to develop CRISPR as a therapeutic platform. This new Cas9 provides a scaffold to expand our Cas9 repertoire, and help us create better models of disease, identify mechanisms, and develop new treatments.”

About the engineered CRISPR-Cas9 system

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) have recently been harnessed as genome editing tools in a wide range of species. The engineered CRISPR-Cas9 system allows researchers to mutate or change the expression of genes in living cells, including those of humans. The family of Cas9 nucleases (also known as Cas5, Csn1, or Csx12) recognizes DNA targets in complex with RNA guides. Researchers can now harness the engineered system to home in on specific nucleic acid sequences and cut the DNA at those precise targets. The cuts modify the activity of the targeted genes, allowing researchers to study the genes’ function.

New way to turn genes on

Anne Trafton |

Using a gene-editing system originally developed to delete specific genes, MIT researchers have now shown that they can reliably turn on any gene of their choosing in living cells.

This new application for the CRISPR/Cas9 gene-editing system should allow scientists to more easily determine the function of individual genes, according to Feng Zhang, 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 the Broad Institute and MIT’s McGovern Institute for Brain Research.

This approach also enables rapid functional screens of the entire genome, allowing scientists to identify genes involved in particular diseases. In a study published in the Dec. 10 online edition of Nature, Zhang and colleagues identified several genes that help melanoma cells become resistant to a cancer drug.

Silvana Konermann, a graduate student in Zhang’s lab, and Mark Brigham, a McGovern Institute postdoc, are the paper’s lead authors.

A new function for CRISPR

The CRISPR system relies on cellular machinery that bacteria use to defend themselves from viral infection. Researchers have previously harnessed this cellular system to create gene-editing complexes that include a DNA-cutting enzyme called Cas9 bound to a short RNA guide strand that is programmed to bind to a specific genome sequence, telling Cas9 where to make its cut.

In the past two years, scientists have developed Cas9 as a tool for turning genes off or replacing them with a different version. In the new study, Zhang and colleagues engineered the Cas9 system to turn genes on, rather than knock them out. Scientists have tried to do this before using proteins that are individually engineered to target DNA at specific sites. However, these proteins are  difficult to work with. “If you use the older generation of tools, getting the technology to do what you actually want is a project on its own,” Konermann says. “It takes a lot of time and is also quite expensive.”

There have also been attempts to use CRISPR to turn on genes by inactivating the part of the Cas9 enzyme that cuts DNA and linking Cas9 to pieces of proteins called activation domains. These domains recruit the cellular machinery necessary to begin reading copying RNA from DNA, a process known as transcription.

However, these efforts have been unable to consistently turn on gene transcription. Zhang and his colleagues, Osamu Nureki and Hiroshi Nishimasu at the University of Tokyo, decided to overhaul the CRISPR-Cas9 system based on an analysis they published earlier this year of the structure formed when Cas9 binds to the guide RNA and its target DNA. “Based on knowing its 3-D shape, we can think about how to rationally improve the system,” Zhang says.

In previous efforts, scientists had tried to attach the activation domains to either end of the Cas9 protein, with limited success. From their structural studies, the MIT team realized that two small loops of the RNA guide poke out from the Cas9 complex and could be better points of attachment because they allow the activation domains to have more flexibility in recruiting transcription machinery.

Using their revamped system, the researchers activated about a dozen genes that had proven difficult or impossible to turn on using the previous generation of Cas9 activators. Each gene showed at least a twofold boost in transcription, and for many genes, the researchers found multiple orders of magnitude increase in activation.

Genome-scale activation screening

Once the researchers had shown that the system was effective at activating genes, they created a library of 70,290 guide RNAs targeting all of the more than 20,000 genes in the human genome.

They screened this library to identify genes that confer resistance to a melanoma drug called PLX-4720. Drugs of this type work well in patients whose melanoma cells have a mutation in the BRAF gene, but cancer cells that survive the treatment can grow into new tumors, allowing the cancer to recur.

To discover the genes that help cells become resistant, the researchers delivered CRISPR components to a large population of melanoma cells grown in the lab, with each cell receiving a different guide RNA targeting a different gene. After treating the cells with PLX-4720, they identified several genes that helped the cells to survive — some previously known to be involved in drug resistance, as well as several novel targets.
Studies like this could help researchers discover new cancer drugs that prevent tumors from becoming resistant.

“You could start with a drug that targets the mutated BRAF along with combination therapy that targets genes that allow the cell to survive. If you target both of them at the same time, you could likely prevent the cells from developing resistance mechanisms that enable further growth despite drug treatment,” Konermann says.

Scientists have tried to do large-scale screens like this by delivering single genes carried by viruses, but that does not work with all genes.

“This new technique could allow you to sample a larger spectrum of genes that might be playing a role,” says Levi Garraway, an associate professor of medicine at Dana-Farber Cancer Institute who was not involved in the research. “This is really a technology development paper, but the tantalizing results from the drug resistance screen speak to the rich biological possibilities of this approach.”

Zhang’s lab also plans to use this technique to screen for genes that, when activated, could correct the effects of autism or neurodegenerative diseases such as Alzheimer’s. He also plans to make the necessary reagents available to academic labs that want to use them, through the Addgene repository.

The research was funded by the National Institute of Mental Health; the National Institute of Neurological Disorders and Stroke; the Keck, Searle Scholars, Klingenstein, Vallee, and Simons foundations; and Bob Metcalfe.

From genes to brains

by Elizabeth Dougherty |

Many brain disorders are strongly influenced by genetics, and researchers have long hoped that the identification of genetic risk factors will provide clues to the causes and possible treatments of these mysterious conditions. In the early years, progress was slow. Many claims failed to replicate, and it became clear that in order to identify the important risk genes with confidence, researchers would need to examine the genomes of very large numbers of patients.

Until recently that would have been prohibitively expensive, but genome research has been accelerating fast. Just how fast was underlined by an announcement in January from a California-based company, Illumina, that it had achieved a long-awaited milestone: sequencing an entire human genome for under $1000. Seven years ago, this task would have cost $10M and taken weeks of work. The new system does the job in a few hours, and can sequence tens of thousands of genomes per year.

In parallel with these spectacular advances, another technological revolution has been unfolding over the past several years, with the development of a new method for editing the genome of living cells. This method, known as CRISPR, allows researchers to make precise changes to a DNA sequence—an advance that is expected to transform many areas of biomedical research and may ultimately form the basis of new treatments for human genetic disease.

The CRISPR technology, which is based on a natural bacterial defense system against viruses, uses a short strand of RNA as a “search string” to locate a corresponding DNA target sequence. This RNA string can be synthesized in the lab and can be designed to recognize any desired sequence of DNA. The RNA carries with it a protein called Cas9, which cuts the target DNA at the chosen location, allowing a new sequence to be inserted—providing researchers with a fast and flexible “search-and-replace” tool for editing the genome.

One of the pioneers in this field is McGovern Investigator Feng Zhang, who along with George Church of Harvard, was the first to show that CRISPR could be used to edit the human genome in living cells. Zhang is using the technology to study human brain disorders, building on the flood of new genetic discoveries that are emerging from advances in DNA sequencing. The Broad Institute, where Zhang holds a joint appointment, is a world leader in human psychiatric genetics, and will be among the first to acquire the new Illumina sequencing machines when they reach the market later this year.

By sequencing many thousands of individuals, geneticists are identifying the rare genetic variants that contribute to risk of diseases such as autism, schizophrenia and bipolar disorder. CRISPR will allow neuroscientists to study those gene variants in cells and in animal models. The goal, says McGovern Institute director Bob Desimone, is to understand the biological roots of brain disorders. “The biggest obstacle to new treatments has been our ignorance of fundamental mechanisms. But with these new technologies, we have a real opportunity to understand what’s wrong at the level of cells and circuits, and to identify the pressure points at which therapeutic intervention may be possible.”

Culture Club

In other fields, the influence of genetic variations on disease has turned out to be surprisingly difficult to unravel, and for neuropsychiatric disease, the challenge may be even greater. The brain is the most complex organ of the body, and the underlying pathologies that lead to disease are not yet well understood. Moreover, any given disorder may show a wide variation in symptoms from patient to patient, and it may also have many different genetic causes. “There are hundreds of genes that can contribute to autism or schizophrenia,” says McGovern Investigator Guoping Feng, who is also Poitras Professor of Neuroscience.

To study these genes, Feng and collaborators at the Broad Institute’s Stanley Center for Psychiatric Research are planning to screen thousands of cultures of neurons, grown in the tiny wells of cell culture plates. The neurons, which are grown from stem cells, can be engineered using CRISPR to contain the genetic variants that are linked to neuropsychiatric disease. Each culture will contain neurons with a different variant, and these will be examined for abnormalities that might be associated with disease.

Feng and colleagues hope this high-throughput platform will allow them to identify cellular traits, or phenotypes, that may be related to disease and which can then be studied in animal models to see if they cause defects in brain function or in behavior. In the longer term, this high-throughput platform can also be used to screen for new drugs that can reverse these defects.

Animal Kingdom

Cell cultures are necessary for large-scale screens, but ultimately the results must be translated into the context of brain circuits and behavior. “That means we must study animal models too,” says Feng.

Feng has created several mouse models of human brain disease by mutating genes that are linked to these disorders and examining the behavioral and cellular defects in the mutant animals. “We have models of obsessive-compulsive disorder and autism,” he explains. “By studying these mice we want to learn what’s wrong with their brains.”

So far, Feng has focused on single-gene models, but the majority of human psychiatric disorders are triggered by multiple genes acting in combination. One advantage of the new CRISPR method is that it allows researchers to introduce several mutations in parallel, and Zhang’s lab is now working to create autistic mice with more than one gene alteration.

Perhaps the most important advantage of CRISPR is that it can be applied to any species. Currently, almost all genetic modeling of human disease is restricted to mice. But while mouse models are convenient, they are limited, especially for diseases that affect higher brain functions and for which there are no clear parallels in rodents. “We also need to study species that are closer to humans,” says Feng.

Accordingly, he and Zhang are collaborating with colleagues in Oregon and China to use CRISPR to create primate models of neuropsychiatric disorders. Earlier this year, a team in China announced that they had used CRISPR to create transgenic monkeys that will be used to study defects in metabolism and immunity.

Feng and Zhang are planning to use a similar approach to study brain disorders, but in addition to macacques, they will also work with a smaller primate species, the marmoset. These animals, with their fast breeding cycles and complex behavioral repertoires, are ideal for genetic studies of behavior and brain function. And because they are very social with highly structured communication patterns, they represent a promising new model for understanding the neural basis of social cognition and its disruption in conditions such as autism.

Given their close evolutionary relationship to humans, marmoset models could also help accelerate the development of new therapies. Many experimental drugs for brain disorders have been tested successfully in mice, only to prove ineffective in subsequent human trials. These failures, which can be enormously expensive, have led many drug companies to cut back on their neuroscience R&D programs. Better animal models could reverse this trend by allowing companies to predict more accurately which drug candidates are most promising, before investing heavily in human clinical trials.

Feng’s mouse research provides an example of how this approach can work. He previously developed a mouse model of obsessive-compulsive disorder, in which the animals engage in obsessive self-grooming, and he has now shown that this effect can be reversed when the missing gene is reintroduced, even in adulthood. Other researchers have seemed similar results with other brain disorders such as Rett Syndrome, a condition that is often accompanied by autism. “The brain is amazingly plastic,” says Feng. “At least in a mouse, we have shown that the damage can often be repaired. If we can also show this in marmosets or other primate models, that would really give us hope that something similar is possible in humans.”

Human Race

Ultimately, to understand the genetic roots of human behavior, researchers must sequence the genomes of individual subjects in parallel with measurements of those same individuals’ behavior and brain function.

Such studies typically require very large sample sizes, but the plummeting cost of sequencing is now making this feasible. In China, for instance, a project is already underway to sequence the genomes of many thousands of individuals to uncover genetic influences on cognition and intelligence.

The next step will be to link the genetics to brain activity, says McGovern Investigator John Gabrieli, who also directs the Martinos Imaging Center at MIT. “It’s a big step to go from DNA to behavioral variation or clinical diagnosis. But we know those genes must affect brain function, so neuroimaging may help us to bridge that gap.”

But brain scans can be time-consuming, given that volunteers must perform behavioral tasks in the scanner. Studies are typically limited to a few dozen subjects, not enough to detect the often subtle effects of genomic variation.

One way to enlarge these studies, says Gabrieli, is to image the brain during rest rather than in a state of prompted activity. This procedure is fast and easy to replicate from lab to lab, and patterns of resting state activity have turned out to be surprisingly reproducible; moreover, Gabrieli is finding that differences in resting activity are associated with brain disorders such as autism, and he hopes that in the future it will be possible to relate these differences to the genetic factors that are emerging from genome studies at the Broad Institute and elsewhere.

“I’m optimistic that we’re going to see dramatic advances in our understanding of neuropsychiatric disease over the next few years.” — Bob Desimone

Confirming these associations will require a “big data” approach, in which results from multiple labs are consolidated into large repositories and analyzed for significant associations. Resting state imaging lends itself to this approach, says Gabrieli. “To find the links between brain function and genetics, big data is the direction we need to go to be successful.”

How soon might this happen? “It won’t happen overnight,” cautions Desimone. “There are a lot of dots that need to be connected. But we’ve seen in the case of genome research how fast things can move once the right technologies are in place. I’m optimistic that we’re going to see equally dramatic advances in our understanding of neuropsychiatric disease over the next few years.”

Genome Editing with CRISPR – Cas9

Anne Trafton |

This animation depicts the CRISPR-Cas9 method for genome editing – a powerful new technology with many applications in biomedical research, including the potential to treat human genetic disease. Feng Zhang, a leader in the development of this technology, is a faculty member at MIT, an investigator at the McGovern Institute for Brain Research, and a core member of the Broad Institute.

 

Feng Zhang shares Gabbay Award for CRISPR research

Anne Trafton |

Feng Zhang of MIT and the Broad Institute, Jennifer Doudna of the University of California, Berkeley and the Howard Hughes Medical Institute, and Emmanuelle Charpentier of Umeå University have been awarded Brandeis University’s 17th Annual Jacob Heskel Gabbay Award in Biotechnology and Medicine.

The researchers are being honored for their work on the CRISPR/cas system, a genome editing technology that allows scientists to make precise changes to a DNA sequence — an advance that is expected to transform many areas of biomedical research and may ultimately form the basis of new treatments for human genetic disease.

Feng Zhang wins NSF’s Alan T. Waterman Award

Anne Trafton |

The National Science Foundation (NSF) named Feng Zhang the 2014 recipient of its Alan T. Waterman Award. This award is NSF’s highest honor that annually recognizes an outstanding researcher under the age of 35 and funds his or her research in any field of science or engineering. Zhang’s research focuses on understanding how the brain works.

“It is a great pleasure to honor Feng Zhang with this award for his young, impressive career,” said NSF Director France Córdova. “It is exciting to support his continued fundamental research, which is certain to impact the field of brain research. Imagine a future free of schizophrenia, autism and other brain disorders that wreak havoc on individuals, families and society. Feng’s research moves us in that direction.”

Zhang seeks to understand the molecular machinery of brain cells through the development and application of innovative technologies. He created and is continuing to perfect tools that afford researchers precise control over biological activities occurring inside the cell. With these tools, researchers can deepen their understanding of how the genome works, and how it influences the development and function of the brain. Zhang also examines failures within the systems that cause disease.

Two different lines of fundamental research and technology development are helping him do that: optogenetics and genome engineering. With Edward Boyden and Karl Deisseroth at Stanford University, he developed optogenetics to study brain circuits, a technique in which light is used to affect signaling and gene expression of neurons involved in complex behaviors. Zhang also developed the CRISPR system to enable new, cheaper, more effective ways to “edit” animal genomes–that is, to identify and cut a short DNA sequence underlying a disorder so that it may be deleted or substituted out for other genetic material. Although Zhang’s main area of focus is the brain, the potential applications of CRISPR technology extend well beyond neuroscience.

“This is an immensely exciting time for the field because of the tremendous potential of tools like CRISPR, which allows us to modify the genomes of mammalian cells,” Zhang said. “One of my long-term goals is to better understand the molecular mechanisms of brain function and identify new ways to treat devastating neurological disorders.”

Since high school, Zhang has devoted his time, energy and intellectual prowess to developing ways to study and repair the nervous system. Today, he is one of 11 core faculty members at the Broad Institute of MIT and Harvard; an investigator at MIT’s McGovern Institute for Brain Research; and the W. M. Keck Career Development Professor with a joint appointment in MIT’s Departments of Brain and Cognitive Sciences and Biological Engineering.

Zhang is widely recognized for his pioneering work in optogenetics and genome editing. He shared the Perl/UNC Neuroscience Prize with Karl Deisseroth and Edward Boyden in 2012. In 2013, MIT Technology Review recognized him as a “pioneer” and one of its 35 Innovators Under 35; Popular Science magazine placed Zhang on its Brilliant 10, an annual list of the most promising scientific innovators. Nature also named him as one of the “ten people who mattered” in 2013 for his work on developing the CRISPR system for genome editing in mammalian cells.

The Waterman award will be presented to Zhang at an evening ceremony at the U.S. Department of State in Washington, D.C., on May 6. At that event, the National Science Board will also present its 2014 Vannevar Bush award to mathematician Richard Tapia and Public Service awards to bioethicist Arthur Caplan and to the AAAS Science & Technology Policy Fellowships Program.

Plans are underway for Zhang to deliver a lecture at a meeting of the National Science Board at NSF and to meet with students at Thomas Jefferson High School for Science and Technology during his visit this spring.