Scientists harness human protein to deliver molecular medicines to cells

Researchers from MIT, the McGovern Institute for Brain Research at MIT, the Howard Hughes Medical Institute, and the Broad Institute of MIT and Harvard have developed a new way to deliver molecular therapies to cells. The system, called SEND, can be programmed to encapsulate and deliver different RNA cargoes. SEND harnesses natural proteins in the body that form virus-like particles and bind RNA, and it may provoke less of an immune response than other delivery approaches.

The new delivery platform works efficiently in cell models, and, with further development, could open up a new class of delivery methods for a wide range of molecular medicines — including those for gene editing and gene replacement. Existing delivery vehicles for these therapeutics can be inefficient and randomly integrate into the genome of cells, and some can stimulate unwanted immune reactions. SEND has the promise to overcome these limitations, which could open up new opportunities to deploy molecular medicine.

“The biomedical community has been developing powerful molecular therapeutics, but delivering them to cells in a precise and efficient way is challenging,” said CRISPR pioneer Feng Zhang, senior author on the study, core institute member at the Broad Institute, investigator at the McGovern Institute, and the James and Patricia Poitras Professor of Neuroscience at MIT. “SEND has the potential to overcome these challenges.” Zhang is also an investigator at the Howard Hughes Medical Institute and a professor in MIT’s Departments of Brain and Cognitive Sciences and Biological Engineering.

SEND packages are introduced to diseased cells to deliver therapeutic mRNA and restore health. Image: McGovern Institute

Reporting in Science, the team describes how SEND (Selective Endogenous eNcapsidation for cellular Delivery) takes advantage of molecules made by human cells. At the center of SEND is a protein called PEG10, which normally binds to its own mRNA and forms a spherical protective capsule around it. In their study, the team engineered PEG10 to selectively package and deliver other RNA. The scientists used SEND to deliver the CRISPR-Cas9 gene editing system to mouse and human cells to edit targeted genes.

First author Michael Segel, a postdoctoral researcher in Zhang’s lab, and Blake Lash, second author and a graduate student in the lab, said PEG10 is not unique in its ability to transfer RNA. “That’s what’s so exciting,” said Segel. “This study shows that there are probably other RNA transfer systems in the human body that can also be harnessed for therapeutic purposes. It also raises some really fascinating questions about what the natural roles of these proteins might be.”

Inspiration from within

The PEG10 protein exists naturally in humans and is derived from a “retrotransposon” — a virus-like genetic element — that integrated itself into the genome of human ancestors millions of years ago. Over time, PEG10 has been co-opted by the body to become part of the repertoire of proteins important for life.

Four years ago, researchers showed that another retrotransposon-derived protein, ARC, forms virus-like structures and is involved in transferring RNA between cells. Although these studies suggested that it might be possible to engineer retrotransposon proteins as a delivery platform, scientists had not successfully harnessed these proteins to package and deliver specific RNA cargoes in mammalian cells.

Knowing that some retrotransposon-derived proteins are able to bind and package molecular cargo, Zhang’s team turned to these proteins as possible delivery vehicles. They systematically searched through these proteins in the human genome for ones that could form protective capsules. In their initial analysis, the team found 48 human genes encoding proteins that might have that ability. Of these, 19 candidate proteins were present in both mice and humans. In the cell line the team studied, PEG10 stood out as an efficient shuttle; the cells released significantly more PEG10 particles than any other protein tested. The PEG10 particles also mostly contained their own mRNA, suggesting that PEG10 might be able to package specific RNA molecules.

Developing a modular system

To develop the SEND technology, the team identified the molecular sequences, or “signals,” in PEG10’s mRNA that PEG10 recognizes and uses to package its mRNA. The researchers then used these signals to engineer both PEG10 and other RNA cargo so that PEG10 could selectively package those RNAs. Next, the team decorated the PEG10 capsules with additional proteins, called “fusogens,” that are found on the surface of cells and help them fuse together.

By engineering the fusogens on the PEG10 capsules, researchers should be able to target the capsule to a particular kind of cell, tissue, or organ. As a first step towards this goal, the team used two different fusogens, including one found in the human body, to enable delivery of SEND cargo.

“By mixing and matching different components in the SEND system, we believe that it will provide a modular platform for developing therapeutics for different diseases,” said Zhang.

Advancing gene therapy

SEND is composed of proteins that are produced naturally in the body, which means it may not trigger an immune response. If this is demonstrated in further studies, the researchers say SEND could open up opportunities to deliver gene therapies repeatedly with minimal side effects. “The SEND technology will complement viral delivery vectors and lipid nanoparticles to further expand the toolbox of ways to deliver gene and editing therapies to cells,” said Lash.

Next, the team will test SEND in animals and further engineer the system to deliver cargo to a variety of tissues and cells. They will also continue to probe the natural diversity of these systems in the human body to identify other components that can be added to the SEND platform.

“We’re excited to keep pushing this approach forward,” said Zhang. “The realization that we can use PEG10, and most likely other proteins, to engineer a delivery pathway in the human body to package and deliver new RNA and other potential therapies is a really powerful concept.”

This work was made possible with support from the Simons Center for the Social Brain at MIT; National Institutes of Health Intramural Research Program; National Institutes of Health grants 1R01-HG009761 and 1DP1-HL141201; Howard Hughes Medical Institute; Open Philanthropy; G. Harold and Leila Y. Mathers Charitable Foundation; Edward Mallinckrodt, Jr. Foundation; Poitras Center for Psychiatric Disorders Research at MIT; Hock E. Tan and K. Lisa Yang Center for Autism Research at MIT; Yang-Tan Center for Molecular Therapeutics at MIT; Lisa Yang; Phillips family; R. Metcalfe; and J. and P. Poitras.

Scientists engineer new CRISPR platform for DNA targeting

A team that includes the scientist who first harnessed the revolutionary CRISPR-Cas9 and other systems for genome editing of eukaryotic organisms, including animals and plants, has engineered another CRISPR system, called Cas12b. The new system offers improved capabilities and options when compared to CRISPR-Cas9 systems.

In a study published today in Nature Communications, Feng Zhang and colleagues at the Broad Institute of MIT and Harvard and the McGovern Institute for Brain Research at MIT, with co-author Eugene Koonin at the National Institutes of Health, demonstrate that the new enzyme can be engineered to target and precisely nick or edit the genomes of human cells. The high target specificity and small size of Cas12b from Bacillus hisashii (BhCas12b) as compared to Cas9 (SpCas9), makes this new system suitable for in vivo applications. The team is now making CRISPR-Cas12b widely available for research.

The team previously identified Cas12b (then known as C2c1) as one of three promising new CRISPR enzymes in 2015, but faced a hurdle: Because Cas12b comes from thermophilic bacteria — which live in hot environments such as geysers, hot springs, volcanoes, and deep sea hydrothermal vents — the enzyme naturally only works at temperatures higher than human body temperature.

“We searched for inspirations from nature,” Zhang said. “We wanted to create a version of Cas12b that could operate at lower temperatures, so we scanned thousands of bacterial genetic sequences, looking in bacteria that could thrive in the lower temperatures of mammalian environments.”

Through a combination of exploration of natural diversity and rational engineering of promising candidate enzymes, they generated a version of Cas12b capable of efficiently editing genomes in primary human T cells, an important initial step for therapeutics that target or leverage the immune system.

“This is further evidence that there are many useful CRISPR systems waiting to be discovered,” said Jonathan Strecker, a postdoctoral fellow in the Zhang Lab, a Human Frontiers Science program fellow, and the study’s first author.

The field is moving quickly: Since the Cas12b family of enzymes was first described in 2015 and demonstrated to be RNA-guided DNA endonucleases, several groups have have been exploring this family of enzymes. In 2017 a team from Jennifer Doudna’s lab at UC Berkeley reported that Cas12b from Alicyclobacillus acidoterrestris can mediate non-specific collateral cleavage of DNA in vitro. More recently, a team from the Chinese Academy of Sciences in Beijing reported that another Cas12b, from Alicyclobacillus acidiphilus, was used to edit mammalian cells.

The Broad Institute and MIT are sharing the Cas12b system widely. As with earlier genome editing tools, these groups will make the 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 reagents more than 52,000 times with researchers at nearly 2,400 labs in 62 countries to accelerate research.

Zhang is a core institute member of the Broad Institute of MIT and Harvard, as well as an investigator at the McGovern Institute for Brain Research at MIT, the James and Patricia Poitras Professor of Neuroscience at MIT, and an associate professor at MIT, with joint appointments in the departments of Brain and Cognitive Sciences and Biological Engineering.

Support for this study was provided by the Poitras Center for Psychiatric Disorders Research, the Hock E. Tan and K. Lisa Yang Center for Autism Research, the National Human Genome Research Institute, the National Institute of Mental Health, the National Heart, Lung, and Blood Institute, and other sources. Feng Zhang is an Investigator with the Howard Hughes Medical Institute.


Strecker J, et al. Engineering of CRISPR-Cas12b for human genome editing. Nature Communications. Online January 22, 2019. DOI: 10.1038/s41467-018-08224-4.

Scientists unveil CRISPR-based diagnostic platform

A team of scientists from the Broad Institute of MIT and Harvard, the McGovern Institute for Brain Research at MIT, the Institute for Medical Engineering and Science at MIT, and the Wyss Institute for Biologically Inspired Engineering at Harvard University has adapted a CRISPR protein that targets RNA (rather than DNA), for use as a rapid, inexpensive, highly sensitive diagnostic tool with the potential to transform research and global public health.

In a study published today in Science, Broad Institute members Feng Zhang, Jim Collins, Deb Hung, Aviv Regev, and Pardis Sabeti describe how this RNA-targeting CRISPR enzyme was harnessed as a highly sensitive detector — able to indicate the presence of as little as a single molecule of a target RNA or DNA. Co-first authors Omar Abudayyeh and Jonathan Gootenberg, graduate students at MIT and Harvard, respectively, dubbed the new tool SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing); this technology could one day be used to respond to viral and bacterial outbreaks, monitor antibiotic resistance, and detect cancer.

The scientists demonstrate the method’s versatility on a range of applications, including:

• detecting the presence of Zika virus in patient blood or urine samples within hours;
• distinguishing between the genetic sequences of African and American strains of Zika virus;
• discriminating specific types of bacteria, such as E. coli;
• detecting antibiotic resistance genes;
• identifying cancerous mutations in simulated cell-free DNA fragments; and
• rapidly reading human genetic information, such as risk of heart disease, from a saliva sample.

Because the tool can be designed for use as a paper-based test that does not require refrigeration, the researchers say it is well-suited for fast deployment and widespread use inside and outside of traditional settings — such as at a field hospital during an outbreak, or a rural clinic with limited access to advanced equipment.

“It’s exciting that the Cas13a enzyme, which was originally identified in our collaboration with Eugene Koonin to study the basic biology of bacterial immunity, can be harnessed to achieve such extraordinary sensitivity, which will be powerful for both science and clinical medicine,” says Feng Zhang, core institute member of the Broad Institute, an investigator at the McGovern Institute, and the James and Patricia Poitras ’63 Professor in Neuroscience and associate professor in the departments of Brain and Cognitive Sciences and Biological Engineering at MIT.

In June 2016, Zhang and his colleagues first characterized the RNA-targeting CRISPR enzyme, now called Cas13a (previously known as C2c2), which can be programmed to cleave particular RNA sequences in bacterial cells. Unlike DNA-targeting CRISPR enzymes (such as Cas9 and Cpf1), Cas13a can remain active after cutting its intended RNA target and may continue to cut other nontargeted RNAs in a burst of activity that Zhang lab scientists referred to as “collateral cleavage.” In their paper and patent filing, the team described a wide range of biotechnological applications for the system, including harnessing RNA cleavage and collateral activity for basic research, diagnostics, and therapeutics.

In a paper in Nature in September 2016, Jennifer Doudna, Alexandra East-Seletsky, and their colleagues at the University of California at Berkeley employed the Cas13a collateral cleavage activity for RNA detection. That method required the presence of many millions of molecules, however, and therefore lacked the sensitivity required for many research and clinical applications.

The method reported today is a million-fold more sensitive. This increase was the result of a collaboration between Zhang and his team and Broad Institute member Jim Collins, who had been working on diagnostics for Zika virus.

Working together, the Zhang and Collins teams were able to use a different amplification process, relying on body heat, to boost the levels of DNA or RNA in their test samples. Once the level was increased, the team applied a second amplification step to convert the DNA to RNA, which enabled them to increase the sensitivity of the RNA-targeting CRISPR by a millionfold, all with a tool that can be used in nearly any setting.

“We can now effectively and readily make sensors for any nucleic acid, which is incredibly powerful when you think of diagnostics and research applications,” says Collins, the Termeer Professor of Medical Engineering and Science at MIT and core faculty member at the Wyss Institute. “This tool offers the sensitivity that could detect an extremely small amount of cancer DNA in a patient’s blood sample, for example, which would help researchers understand how cancer mutates over time. For public health, it could help researchers monitor the frequency of antibiotic-resistant bacteria in a population. The scientific possibilities get very exciting very quickly.”

One of the most urgent and obvious applications for this new diagnostic tool would be as a rapid, point-of-care diagnostic for infectious disease outbreaks in resource-poor areas.
“There is great excitement around this system,” says Deb Hung, co-author and co-director of the Broad’s Infectious Disease and Microbiome Program. “There is still much work to be done, but if SHERLOCK can be developed to its full potential it could fundamentally change the diagnosis of common and emerging infectious diseases.”

“One thing that’s especially powerful about SHERLOCK is its ability to start testing without a lot of complicated and time-consuming upstream experimental work,” says Pardis Sabeti, also a co-author in the paper. In the wake of the ongoing Zika outbreak, Sabeti and the members of her lab have been working to collect samples, rapidly sequence genomes, and share data in order to accelerate the outbreak response effort. “This ability to take raw samples and immediately start processing could transform the diagnosis of Zika and a boundless number of other infectious diseases,” she says. “This is just the beginning.”

Additional authors include Jeong Wook Lee, Patrick Essletzbichler, Aaron J. Dy, Julia Joung, Vanessa Verdine, Nina Donghia, Nichole M. Daringer, Catherine A. Freije, Cameron Myhrvold, Roby P. Bhattacharyya, Jonathan Livny, and Eugene V. Koonin.

Reading the rules of gene regulation

We have a reasonable understanding of the rules behind the genome’s protein-coding components. We can look at a DNA sequence and point with confidence to where a gene’s coding region begins, where it ends, and pieces of its geography.

For the remaining 98 percent of the genome — the part that dictates which genes a cell reads — it’s a different story. What knowledge we have of the rules governing this “dark matter” comes from from studying and manipulating individual bits of noncoding DNA one at a time. The rulebook that governs how the noncoding genome works, however, has remained out of reach.

“Ninety percent of the genetic variations that affect human disease are in the noncoding regions,” said Broad founding director Eric Lander. “But we haven’t had any way to tell, in a systematic way, which regulators affect which genes.”

In a pair of newly published Science papers, two research teams at the Broad show how methods leveraging CRISPR gene editing could help grasp those rules.

Using two complementary approaches, the teams — one from the Lander lab, the other from that of Broad Core Institute Member and McGovern Institute for Brain Research investigator Feng Zhang — used CRISPR as a tool to systematically probe thousands of noncoding DNA sequences simultaneously (much as Zhang and others did previously with coding DNA). In the process, both identified several interesting genetic regulators, including ones millions of bases away from the genes they control.

“We’d like to be able to catalog the noncoding elements that control every gene’s expression in every cell type,” said Jesse Engreitz, a postdoctoral fellow in the Lander lab and senior author on one of the papers. “This is a massive problem in biology, and it’s a rate-limiting step for connecting many genetic associations to their fundamental molecular mechanisms and to human disease.”
Variations on a theme

Both teams used pooled CRISPR screens (which scan and edit large swaths of the genome simultaneously using a molecular scalpel called the Cas9 enzyme and thousands of guide RNAs, which target Cas9 to specific sequences) to perturb noncoding DNA. But they did so in different ways.

Zhang, Neville Sanjana (a Zhang lab alum and now a core member of the New York Genome Center), and Jason Wright (another Zhang alum, now at Homology Medicines) used Cas9 to make precise edits to overlapping stretches of noncoding DNA — in their case, in regions surrounding three genes (NF1, NF2, and CUL3) whose functional loss has been linked to drug resistance in a form of melanoma.

“This approach lets us induce a wide diversity of mutations,” Sanjana explained. “We don’t have to speculate how a given sequence might best be disrupted.”

Engreitz, Lander, and graduate student Charles Fulco, on the other hand, employed a CRISPR interference system, using an inactive or “dead” form of Cas9 fused to a protein fragment called a KRAB domain to silence their target sequences (around MYC and GATA1, the genes for two important transcription factors).

“This system provides a good quantitative estimate of a given noncoding region’s regulatory influence,” Engreitz said. “It both shows you where the dials are that control a given gene, and tells you how much each dial matters.”

Each team then used a functional readout (increased drug resistance in melanoma cells for Sanjana, Wright, and Zhang; a drop in cell growth for Fulco, Lander, and Engreitz) and deep sequencing to see which of their guide RNAs impacted expression of their genes of interest and map the regulators those guide RNAs affected.

The two teams’ findings, confirmed with an array of additional techniques (e.g., chromatin profiling, 3D conformational capture, transcription factor profiling), point to the potential for tracing the noncoding genome’s regulatory wiring leveraging CRISPR tools. Fulco, Lander, and Engreitz found and ranked the relative importance of seven MYC and three GATA1 enhancers (short pieces of noncoding DNA that boost a gene’s chances of being read). Sanjana, Wright, and Zhang’s screen pinpointed numerous enhancers and transcription factor binding sites just for CUL3 alone.
Studying sequences in their natural habitat

While similar in principle to traditional reporter assays (where scientists couple interesting sequences to reporter genes in plasmids), these pooled CRISPR screens have a distinct difference: they probe the sequences directly, in their native habitat.

“The screens interrogate the sequences in their endogenous context,” Sanjana emphasized. “Reporter assays can be very helpful, but they lack the 3D conformation or local chromatin environment of the native genomic context. Here, the regulatory sequences undergo all of their normal interactions.”

“For example, we could see long-range loops between gene promoters and noncoding sites thousands of bases away,” he continued. “We would have missed these interesting 3D interactions entirely if we just looked at these regulatory elements in isolation.”

One limitation, Engreitz noted, is that neither CRISPR approach, in its current form, addresses the genome’s inherent redundancy. “Maybe it’s not enough to break one enhancer to really understand how a gene is controlled. Maybe you have to break more than one,” he said. “We can’t do that yet.”

But Engreitz, Sanjana, and Lander are all optimistic about the potential for using CRISPR-based approaches to reveal the noncoding genome’s underlying order.

“One interesting challenge with the noncoding genome is that while it is huge, the individual functional elements within it can be quite small,” Sanjana said. “In the future, it will be important to think about how we can develop new approaches that interrogate larger regions while maintaining high resolution.”

Engreitz agreed, adding, “There’s a potential that as we map more of these connections we’re going to learn the rules that let us predict them for the rest of the noncoding genome.”

“These approaches, using libraries of guide RNAs to bring CRISPR in to cut or bring in inhibitors, let you directly see the effects of large areas of noncoding DNA on different genes,” Lander said. “I think this is going to crack open systematic maps of gene regulation.”

Papers cited:

Fulco CP et al. Systematic mapping of functional enhancer-promoter connections with CRISPR interference. Science. September 29, 2016. DOI: 10.1126/science.aag2445

Sanjana NE et al. High-resolution interrogation of functional elements in the noncoding genome. Science. September 29, 2016. DOI: 10.1126/science.aaf7613

Divide and conquer

Cell populations are remarkably diverse—even within the same tissue or cell type. Each cell, no matter how similar it appears to its neighbor, behaves and responds to its environment in its own way depending on which of its genes are expressed and to what degree. How genes are expressed in each cell—how RNA is “read” and turned into proteins—determines what jobs the cell performs in the body.

Traditionally, researchers have taken an en masse approach to studying gene expression, extracting an averaged measurement derived from an entire cell population. But over the past few years, single cell sequencing has emerged as a transformative tool, enabling scientists to look at gene expression within cells at an unprecedented resolution. With single-cell technologies, researchers have been able to examine the heterogeneity within cell populations; identify rare cells; observe interactions between diverse cell types; and better understand how these interactions influence health and disease.

This week in Science, researchers from the labs of Broad core institute members Aviv Regev and Feng Zhang, of MIT and MIT’s McGovern Institute respectively, report on their newest contribution to this field: Div-Seq, a method that enables the study of previously intractable and rare cell types in the brain. The study’s first authors, Naomi Habib, a postdoctoral fellow in the Regev and Zhang labs, and Yinqing Li, also a postdoc in the Zhang lab, sat down to answer questions about this groundbreaking approach.

Why is it so important to study neurons at the single cell level?

Li: Neuropsychiatric diseases are often too complex to find an effective treatment, partly because the neurons, that underly the disease are heterogeneous. Only when we have a full atlas of every neuron type at single-cell resolution—and figure out which ones are the cause of the pathology—can we develop a targeted and effective therapy. With this goal in mind, we developed sNuc-Seq and Div-Seq to make it technologically possible to profile neurons from the adult brain at significantly improved resolution, fidelity, and sensitivity.

Scientifically, what was the need that you were trying to address when you started this study?

Habib: Going into this study we were specifically interested in studying so-called “newborn” neurons, which are rare and hard to find. We think of our brain as being non-regenerative, but in fact there are rare, neuronal stem cells in specific areas of the brain that divide and create new neurons throughout our lives. We wanted to understand how gene expression changed as these cells developed. Typically when people studied gene expression in the brain they just mashed up tissue and took average measurements from that mixture. Such “bulk” measurements are hard to interpret and we lose the gene expression signals that come from individual cell types.

When I joined the Zhang and Regev labs, some of the first single cell papers were coming out, and it seemed like the perfect approach for advancing the way we do neuroscience research; we could measure RNA at the single cell level and really understand what different cell types were there, including rare cells, and what they contribute to different brain functions. But there was a problem. Neurons do not look like regular cells: they are intricately connected. In the process of separating them, the cells do not stay intact and their RNA gets damaged, and this problem increases with age.

So what was your solution?

Habib: Isolating single neurons is problematic, but the nucleus is nice and round and relatively easy to isolate. That led us to ask, “Why not try single nucleus RNA sequencing instead of single cell sequencing?” We called it “sNuc-Seq.”

It worked well. We get a lot of information from the RNA in the nucleus; we can learn what cell type we’re looking at, what state of development it’s in, and what kind of processes are going on in the cell—all of the key information we would want to get from RNA sequencing.

Then, to make it possible to find the rare newborn neurons, we developed Div-Seq. It’s based on sNuc-Seq, but we introduce a compound that incorporates into DNA and labels the DNA while it’s replicating, so it’s specific for newly divided cells. Because we already isolated the nuclei, it’s fairly simple from there to fluorescently tag the labeled cells, sort them, and get RNA for sequencing.

You tested this method while preparing your paper. What did you find?

Habib: We studied “newborn” neurons from the brain across multiple time-points. We could see the changes in gene expression that occur throughout adult neurogenesis; the cells transition from state-to-state—from stem cells to mature neurons—and during these transitions, we found a coordinated change in the expression of hundreds of genes. It was beautiful to see these signatures, and they enabled us to pinpoint regulatory genes expressed during specific points of the cell differentiation process.

We were also able to look at where regeneration occurs. We decided to look in the spinal cord because there is a lot of interest in understanding the potential of regeneration to help with spinal cord injury. Div-Seq enabled us to scan millions of neurons and isolate the small percentage that were dividing and characterize each by its RNA signature. We found that within the spinal cord there is ongoing regeneration of a specific type of neuron—GABAergic neurons. That was an exciting finding that also showed the utility of our method.

Are the data you get from this method compatible with data from previous single-cell techniques?

Li: Because this method is specifically designed to address the particular challenges of profiling neurons, the data from this method is distinct from that obtained from previous single-cell techniques. Since the data was new to this approach, a novel computational tool was developed in this project in order to fully reveal the rich information, which is now available to the scientific community.

Are there other benefits of using this method?

Habib: Single nucleus RNA-seq enables the study of the adult and aging brain at the single cell level, which is now being applied to study cellular diversity across the brain during health and disease. Our approach also makes it easier to explore any complex tissue where single cells are hard to obtain for technical reasons. One important aspect is that it works on frozen and fixed tissue, which opens up opportunities to study human samples, such as biopsies, that may be collected overseas or frozen for days or even years.

Additionally, Div-Seq opens new ways to look at the rare process of adult neurogenesis and other regenerative processes that might have been challenging before. Because Div- Seq specifically labels dividing cells, it is a great tool to use to see what cells are dividing in a given tissue and to track gene expression changes over time.

What is the endgame of studying these processes? Can you put this work in context of human health and disease?

Li: We hope that the methods in this study will provide a starting point and method for future work on neuropsychiatric diseases. As we expand our understanding of cell types and their signatures, we can start to ask questions like: Which cells express disease associated genes? Where are these cells located in the brain? What other genes are expressed in these cells, and which might serve as potential drug targets? This approach could help bridge human genetic association studies and molecular neurobiology and open new windows into disease pathology and potential treatments.

Habib: These two methods together enable many applications, which were either very hard or impossible to do before. For example, we characterized the cellular diversity of a region of the brain important for learning and memory—the first region affected in Alzheimer’s disease. Having that understanding—knowing what the normal state of cells is at the molecular level and what went wrong in each individual cell type—can advance our understanding of the disease and perhaps aid in the search for a treatment. We are also excited by the prospect of finding naturally-occurring regeneration in the brain and spine, which could have implications for the field of regenerative medicine in treating, for example, neuronal degeneration or spinal injury.

Paper cited:

Habib N, Li Y, et al. Div-Seq: Single nucleus RNA-Seq reveals dynamics of rare adult newborn neurons. Science. Online July 28, 2016.

Feng Zhang named 2016 Tang Prize Laureate

Feng Zhang, a core institute member of the Broad Institute, an investigator at the McGovern Institute for Brain Research at MIT, and W. M. Keck Career Development Associate Professor in MIT’s Department of Brain and Cognitive Sciences with a joint appointment in Biological Engineering, has been named a 2016 Tang Prize Laureate in Biopharmaceutical Science for his role in developing the CRISPR-Cas9 gene-editing system and demonstrating pioneering uses in eukaryotic cells.

The Tang Prize is a biennial international award granted by judges convened by Academia Sinica, Taiwan’s top academic research institution.

In January 2013 Zhang and his team were first to report CRISPR-based genome editing in mammalian cells, in what has become the most-cited paper in the CRISPR field. Zhang shares the award with Emmanuelle Charpentier of the Max Planck Institute and Jennifer A. Doudna of the University of California at Berkeley.

“To be recognized with the Tang Prize is an incredible honor for our team and it demonstrates the impact of the entire CRISPR field, which began with microbiologists and will continue for years to come as we advance techniques for genome editing,” Zhang said. “Thanks to the scientific community’s commitment to collaboration and an emphasis on sharing across institutions and borders, the last few years have seen a revolution in our ability to understand cancer, autoimmune disease, mental health and infectious disease. We are entering a remarkable period in our understanding of human health.”

Although Zhang is well-known for his work with CRISPR, the 34-year-old scientist has a long track record of innovation. As a graduate student at Stanford University, Zhang worked with Karl Deisseroth and Edward Boyden, who is now also a professor at MIT, to develop optogenetics, in which neuronal activity can be controlled with light. The three shared the Perl-UNC Prize in Neuroscience in 2012 as recognition of these efforts. Zhang has also received the National Science Foundation’s Alan T. Waterman Award (2014), the Jacob Heskel Gabbay Award in Biotechnology and Medicine (2014, shared with Charpentier and Doudna), the Tsuneko & Reiji Okazaki Award (2015), the Human Genome Organization (HUGO) Chen New Investigator Award (2016), and the Canada Gairdner International Award (2016, shared with Charpentier and Doudna, as well as Rodolphe Barrangou from North Carolina State University and Philippe Horvath from DuPont Nutrition & Health).

One of Zhang’s long-term goals is to use genome-editing technologies to better understand the nervous system and develop new approaches to the treatment of neurological and psychiatric diseases. The Zhang lab has shared CRISPR-Cas9 components in response to more than 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. In his current research, he and his students and postdoctoral fellows continue to improve and expand the gene-editing toolbox.

“Professor Zhang’s lab has become a global hub for CRISPR research,” said MIT Provost Martin Schmidt. “His group has shared CRISPR-Cas9 components with tens of thousands of scientists, and has trained many more in the use of CRISPR-Cas9 technology. The Tang Prize is a fitting recognition of all that Professor Zhang has done, and continues to do, to advance this field.”

“CRISPR is a powerful new tool that is transforming biological science while promising revolutionary advances in health care,” said Michael Sipser, dean of the School of Science and Donner Professor of Mathematics at MIT. “We are delighted that Feng Zhang, together with Jennifer Doudna and Emmanuelle Charpentier, have been recognized with the Tang Prize.”

“It is wonderful that the Academia Sinica has chosen to recognize the CRISPR field with this year’s Tang Prize,” said Eric Lander, founding director of the Broad Institute. “On behalf of my colleagues at the Broad and MIT, I wish to congratulate Feng, as well as Emmanuelle Charpentier and Jennifer Doudna, along with the many teams of scientists and all others who have contributed to these transformational discoveries.”

Founded in 2012 by Samuel Yin, the Tang Prize is a non-governmental, non-profit educational foundation that awards outstanding contributions in four fields: sustainable development, biopharmaceutical science, sinology, and rule of law. Nomination and selection of laureates is conducted by the Academia Sinica. Each award cycle, the academy convenes four autonomous selection committees, each consisting of an assembly of international experts, until a consensus on the recipients is reached. Recipients are chosen on the basis of the originality of their work along with their contributions to society, irrespective of nationality, ethnicity, gender, and political affiliation.

This year marks the second awarding of the prize. This year’s awardees will receive the medal, diploma, and cash prize at an award ceremony on September 25 in Taipei. Recipients in each Tang Prize category receive a total of approximately $1.24 million (USD) and a grant of approximately $311,000 (USD). The cash prize and grants are divided equally among joint recipients in each category.


New CRISPR system for targeting RNA

Researchers from MIT and the Broad Institute of MIT and Harvard, as well as the National Institutes of Health, Rutgers University at New Brunswick, and the Skolkovo Institute of Science and Technology, have characterized a new CRISPR system that targets RNA, rather than DNA.

The new approach has the potential to open a powerful avenue in cellular manipulation. Whereas DNA editing makes permanent changes to the genome of a cell, the CRISPR-based RNA-targeting approach may allow researchers to make temporary changes that can be adjusted up or down, and with greater specificity and functionality than existing methods for RNA interference.

In a study published today in Science, Feng Zhang and colleagues at the Broad Institute and the McGovern Institute for Brain Research at MIT, along with co-authors Eugene Koonin and his colleagues at the NIH, and Konstantin Severinov of Rutgers University at New Brunswick and Skoltech, report the identification and functional characterization of C2c2, an RNA-guided enzyme capable of targeting and degrading RNA.

The findings reveal that C2c2 — which is the first naturally occurring CRISPR system known to target only RNA, and was discovered by this collaborative group in October 2015 — helps protect bacteria against viral infection. The researchers demonstrate that C2c2 can be programmed to cleave particular RNA sequences in bacterial cells, which would make it an important addition to the molecular biology toolbox.

The RNA-focused action of C2c2 complements the CRISPR-Cas9 system, which targets DNA, the genomic blueprint for cellular identity and function. The ability to target only RNA, which helps carry out the genomic instructions, offers the ability to specifically manipulate RNA in a high-throughput manner — and to manipulate gene function more broadly. This has the potential to accelerate progress to understand, treat, and prevent disease.

“C2c2 opens the door to an entirely new frontier of powerful CRISPR tools,” said senior author Feng Zhang, who is a core institute member of the Broad Institute, an investigator at the McGovern Institute for Brain Research at MIT, and the W. M. Keck Career Development Associate Professor in MIT’s Department of Brain and Cognitive Sciences.
“There are an immense number of possibilities for C2c2, and we are excited to develop it into a platform for life science research and medicine.”

“The study of C2c2 uncovers a fundamentally novel biological mechanism that bacteria seem to use in their defense against viruses,” said Eugene Koonin, senior author and leader of the Evolutionary Genomics Group at the NIH. “Applications of this strategy could be quite striking.”

Currently, the most common technique for performing gene knockdown is small interfering RNA (siRNA). According to the researchers, C2c2 RNA-editing methods suggest greater specificity and hold the potential for a wider range of applications, such as:

  • Adding modules to specific RNA sequences to alter their function — how they are translated into proteins — which would make them valuable tools for large-scale screens and constructing synthetic regulatory networks; and
  • Harnessing C2c2 to fluorescently tag RNAs as a means to study their trafficking and subcellular localization.

In this work, the team was able to precisely target and remove specific RNA sequences using C2c2, lowering the expression level of the corresponding protein. This suggests C2c2 could represent an alternate approach to siRNA, complementing the specificity and simplicity of CRISPR-based DNA editing and offering researchers adjustable gene “knockdown” capability using RNA.

C2c2 has advantages that make it suitable for tool development:

  • C2c2 is a two-component system, requiring only a single guide RNA to function; and
  • C2c2 is genetically encodable — meaning the necessary components can be synthesized as DNA for delivery into tissue and cells.

“C2c2’s greatest impact may be made on our understanding of the role of RNA in disease and cellular function,” said co-first author Omar Abudayyeh, a graduate student in the Zhang Lab.

Feng Zhang receives 2016 Canada Gairdner International Award

Feng Zhang, a core institute member of the Broad Institute, an investigator at the McGovern Institute for Brain Research at MIT, and W. M. Keck Career Development Associate Professor in MIT’s Department of Brain and Cognitive Sciences, has been named a recipient of the 2016 Canada Gairdner International Award — Canada’s most prestigious scientific prize — for his role in developing the CRISPR-Cas9 gene-editing system.

In January 2013 Zhang and his team were first to report CRISPR-based genome editing in mammalian cells, in what has become the most-cited paper in the CRISPR field. He is one of five scientists the Gairdner Foundation is honoring for work with CRISPR. Zhang shares the award with Rodolphe Barrangou from North Carolina State University; Emmanuelle Charpentier of the Max Planck Institute; Jennifer Doudna of the University of California at Berkeley and Phillipe Horvath from DuPont Nutrition and Health.

“The Gairdner Award is a tremendous recognition for my entire team, and it is a great honor to share this recognition with other pioneers in the CRISPR field,” Zhang says. “In the next decade, the understanding and the discoveries that scientists are going to be able to make using the CRISPR-Cas9 system will lead to new innovations that will translate into new therapeutics and new products that can benefit our lives.”

Although Zhang is well-known for his work with CRISPR, the 34-year-old scientist has a long track record of innovation. As a graduate student at Stanford University, Zhang worked with Karl Deisseroth and Edward Boyden, who is now also a professor at MIT, to develop optogenetics, in which neuronal activity can be controlled with light. The three shared the Perl-UNC Prize in Neuroscience in 2012 as recognition of these efforts. Zhang has also received the National Science Foundation’s Alan T. Waterman Award (2014), the Jacob Heskel Gabbay Award in Biotechnology and Medicine (2014, shared with Charpentier and Doudna), the Tsuneko & Reiji Okazaki Award (2015), and the Human Genome Organization (HUGO) Chen New Investigator Award (2016).

One of Zhang’s long-term goals is to use genome-editing technologies to better understand the nervous system and develop new approaches to the treatment of psychiatric disease. The Zhang lab 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. In his current research, he continues to improve and expand the gene-editing toolbox. “I feel incredibly fortunate and excited to work with an incredible team of students and postdocs to continue advancing our ability to edit and understand the genome,” Zhang says.

“CRISPR is a revolutionary breakthrough that will advance the frontiers of science and enable us to meet the health challenges of the 21st century in ways we are only beginning to imagine,” says Michael Sipser, dean of MIT’s School of Science and the Barton L. Weller Professor of Mathematics. “I am exceedingly proud of the contributions Feng has made to MIT and the greater community of scientists, and extend my heartfelt congratulations to him and his colleagues.”

“CRISPR is a great example of how the scientific community can come together and make stunning progress in a short period of time,” says Eric Lander, founding director of the Broad Institute. “On behalf of my colleagues at the Broad and MIT, I wish to congratulate Feng and all the winners of this prestigious award, as well as the teams of scientists and all others who have contributed to these transformational discoveries.”

The Canada Gairdner International Awards, created in 1959, are given annually to recognize and reward the achievements of medical researchers whose work contributes significantly to the understanding of human biology and disease. The awards provide a $100,000 (CDN) prize to each scientist for their work. Each year, the five honorees of the International Awards are selected after a rigorous two-part review, with the winners voted by secret ballot by a medical advisory board composed of 33 eminent scientists from around the world.

The Broad Institute of MIT and Harvard was launched in 2004 to empower this generation of creative scientists to transform medicine. The Broad Institute seeks to describe all the molecular components of life and their connections; discover the molecular basis of major human diseases; develop effective new approaches to diagnostics and therapeutics; and disseminate discoveries, tools, methods, and data openly to the entire scientific community.

Founded by MIT, Harvard, Harvard-affiliated hospitals, and the visionary Los Angeles philanthropists Eli and Edythe L. Broad, the Broad Institute includes faculty, professional staff, and students from throughout the MIT and Harvard biomedical research communities and beyond, with collaborations spanning over a hundred private and public institutions in more than 40 countries worldwide. For further information about the Broad Institute, visit:

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

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

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.”