Research Area: Genome Engineering

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

CRISPR makes several Discovery of the Decade lists

by Sabbi Lall |

As we reach milestones in time, it’s common to look back and review what we learned. A number of media outlets, including National Geographic, NPR, The Hill, Popular Mechanics, Smithsonian Magazine, Nature, Mental Floss, CNBC, and others, recognized the profound impact of genome editing, adding CRISPR to their discovery of the decade lists.

“In 2013, [CRISPR] was used for genome editing in a eukaryotic cell, forever altering the course of biotechnology and, ultimately our relationship with our DNA.”
— Popular Mechanics

It’s rare for a molecular system to become a household name, but in less than a decade, CRISPR has done just that. McGovern Investigator Feng Zhang played a key role in leveraging CRISPR, an immune system found originally in prokaryotic – bacterial and archaeal – cells, into a broadly customizable toolbox for genomic manipulation in eukaryotic (animal and plant) cells. CRISPR allows scientists to easily and quickly make changes to genomes, has revolutionized the biomedical sciences, and has major implications for control of infectious disease, agriculture, and treatment of genetic disorders.

Shrinking CRISPR tools

by Sabbi Lall |

Before CRISPR gene-editing tools can be used to treat brain disorders, scientists must find safe ways to deliver the tools to the brain. One promising method involves harnessing viruses that are benign, and replacing non-essential genetic cargo with therapeutic CRISPR tools. But there is limited room for additional tools in a vector already stuffed with essential gear.

Squeezing all the tools that are needed to edit the genome into a single delivery vector is a challenge. Soumya Kannan is addressing this capacity problem in Feng Zhang’s lab with fellow graduate student Han Altae-Tran, by developing smaller CRISPR tools that can be more easily packaged into viral vectors for delivery. She is focused on RNA editors, members of the Cas13 family that can fix small mutations in RNA without making changes to the genome itself.

“The limitation is that RNA editors are large. At this point though, we know that editing works, we understand the mechanism by which it works, and there’s feasible packaging in AAV. We’re now trying to shrink systems such as RESCUE and REPAIR so that they fit into the packaging for delivery.”

One of many avenues the Zhang lab has taken to tool-finding in the past is to explore biodiversity for new versions of tools, and this is an approach that intrigues Soumya.

“Metagenomics projects are literally sequencing life from the Antarctic ice cores to hot sea vents. It fascinates me that the CRISPR tools of ancient organisms and those that live in extreme conditions.”

Researchers continue to search these troves of sequencing data for new tools.

 

Two CRISPR scientists on the future of gene editing

by Sabbi Lall |

As part of our Ask the Brain series, Martin Wienisch and Jonathan Wilde of the Feng lab look into the crystal ball to predict the future of CRISPR tech.

_____

Where will CRISPR be in five years?

Jonathan: We’ll definitely have more efficient, more precise, and safer editing tools. An immediate impact on human health may be closer than we think through more nutritious and resilient crops. Also, I think we will have more viable tools available for repairing disease-causing mutations in the brain, which is something that the field is really lacking right now.

Martin: And we can use these technologies with new disease models to help us understand brain disorders such as Huntington’s disease.

Jonathan: There are also incredible tools being discovered in nature: exotic CRISPR systems from newly discovered bacteria and viruses. We could use these to attack disease-causing bacteria.

Martin: We would then be using CRISPR systems for the reason they evolved. Also improved gene drives, CRISPR-systems that can wipe out disease-carrying organisms such as mosquitoes, could impact human health in that time frame.

What will move gene therapy forward?

Martin: A breakthrough on delivery. That’s when therapy will exponentially move forward. Therapy will be tailored to different diseases and disorders, depending on relevant cell types or the location of mutations for example.

Jonathan: Also panning biodiversity even faster: we’ve only looked at one small part of the tree of life for tools. Sequencing and computational advances can help: a future where we collect and analyze genomes in the wild using portable sequencers and laptops can only quicken the pace of new discoveries.

_____

Do you have a question for The Brain? Ask it here.

CRISPR: From toolkit to therapy

by Sabbi Lall |

Think of the human body as a community of cells with specialized roles. Each cell carries the same blueprint, an array of genes comprising the genome, but different cell types have unique functions — immune cells fight invading bacteria, while neurons transmit information.

But when something goes awry, the specialization of these cells becomes a challenge for treatment. For example, neurons lack active cell repair systems required for promising gene editing techniques like CRISPR.

Can current gene editing tools be modified to work in neurons? Can we reach neurons without impacting healthy cells nearby? McGovern Institute researchers are trying to answer these questions by developing gene editing tools and delivery systems that can target — and repair — faulty brain cells.

Expanding the toolkit

Feng Zhang with folded arms in lab
McGovern Investigator Feng Zhang in his lab.

Natural CRISPR systems help bacteria fend off would-be attackers. Our first glimpse of the impact of such systems was the use of CRISPR-Cas9 to edit human cells.

“Harnessing Cas9 was a major game-changer in the life sciences,” explains Feng Zhang, an investigator at the McGovern Institute and the James and Patricia Poitras Professor of Neuroscience at MIT. “But Cas9 is just one flavor of one kind of bacterial defense system — there is a treasure trove of natural systems that may have enormous potential, just waiting to be unlocked.”

By finding and optimizing new molecular tools, the Zhang lab and others have developed CRISPR tools that can now potentially target neurons and fix diverse mutation types, bringing gene therapy within reach.

Precise in space and time

A single letter change to a gene can be devastating. These genes may function only briefly during development, so a temporary “fix” during this window could be beneficial. For such cases, the Zhang lab and others have engineered tools that target short-lived RNAs. These molecules act as messengers, carrying information from DNA to be converted into functional factors in the cell.

“RNA editing is powerful from an ethical and safety standpoint,” explains Soumya Kannan, a graduate student in the Zhang lab working on these tools. “By targeting RNA molecules, which are only present for a short time, we can avoid permanent changes to the genetic material, and we can make these changes in any type of cell.”

Soumya Kannan in the lab
Graduate student Soumya Kannan is developing smaller CRISPR tools that can be more easily packaged into viral vectors for delivery. Photo: Caitlin Cunningham

Zhang’s team has developed twin RNA-editing tools, REPAIR and RESCUE, which can fix single RNA bases by bringing together a base editor with the CRISPR protein Cas13. These RNA-editing tools can be used in neurons because they do not rely on cellular machinery to make the targeted changes. They also have the potential to tackle a wide array of diseases in other tissue types.

CAST addition

If a gene is severely disrupted, more radical help may be needed: insertion of a normal gene. For this situation, Zhang’s lab recently identified CRISPR-associated transposases (CASTs) from cyanobacteria. CASTs combine Cas12k, which is targeted by a guide RNA to a precise genome location, with an enzyme that can insert gene-sized pieces of DNA.

“With traditional CRISPR you can make simple changes, similar to changing a few letters or words in a Word document. The new system can ‘copy and paste’ entire genes.” – Alim Ladha

Transposases were originally identified as enzymes that help rogue genes “jump” from one place to another in the genome. CAST uses a similar activity to insert entire genes self-sufficiently without help from the target cell so, like REPAIR and RESCUE, it can potentially be used in neurons.

“Our initial work was to fully characterize how this new system works, and test whether it can actually insert genes,” explains Alim Ladha, a graduate fellow in the Tan-Yang Center for Autism Research, who worked on CAST with Jonathan Strecker, a postdoctoral fellow in the Zhang lab.

The goal is now to use CAST to precisely target neurons and other specific cell types affected by disease.

Toward delivery

As the gene-editing toolbox expands, McGovern labs are working on precise delivery systems.Adeno-associated virus (AAV) is an FDA-approved virus for delivering genes, but has limited room to carry the necessary cargo — CRISPR machinery plus templates — to fix genes.

To tackle this problem, McGovern Investigators Guoping Feng and Feng Zhang are working on reducing the cargo needed for therapy. In addition, the Zhang, Gootenberg and Abudayyeh labs are working on methods to precisely deliver the therapeutic packages to neurons, such as new tissue-specific viruses that can carry bigger payloads. Finally, entirely new modalities for delivery are being explored in the effort to develop gene therapy to a point where it can be safely delivered to patients.

“Cas9 has been a very useful tool for the life sciences,” says Zhang. “And it’ll be exciting to see continued progress with the broadening toolkit and delivery systems, as we make further progress toward safe gene therapies.

McGovern scientists named STAT Wunderkinds

by Sabbi Lall |

McGovern researchers Sam Rodriques and Jonathan Strecker have been named to the class of 2019 STAT wunderkinds. This group of 22 researchers was selected from a national pool of hundreds of nominees, and aims to recognize trail-blazing scientists that are on the cusp of launching their careers but not yet fully independent.

“We were thrilled to receive this news,” said Robert Desimone, director of the McGovern Institute. “It’s great to see the remarkable progress being made by young scientists in McGovern labs be recognized in this way.”

Finding context

Sam Rodriques works in Ed Boyden’s lab at the McGovern Institute, where he develops new technologies that enable researchers to understand the behaviors of cells within their native spatial and temporal context.

“Psychiatric disease is a huge problem, but only a handful of first-in-class drugs for psychiatric diseases approved since the 1960s,” explains Rodriques, also affiliated with the MIT Media Lab and Broad Institute. “Coming up with novel cures is going to require new ways to generate hypotheses about the biological processes that underpin disease.”

Rodriques also works on several technologies within the Boyden lab, including preserving spatial information in molecular mapping technologies, finding ways of following neural connectivity in the brain, and Implosion Fabrication, or “Imp Fab.” This nanofabrication technology allows objects to be evenly shrunk to the nanoscale and has a wide range of potential applications, including building new miniature devices for examining neural function.

“I was very surprised, not expecting it at all!” explains Rodriques when asked about becoming a STAT Wunderkind, “I’m sure that all of the hundreds of applicants are very accomplished scientists, and so to be chosen like this is really an honor.”

New tools for gene editing

Jonathan Strecker is currently a postdoc working in Feng Zhang’s lab, and associated with both the McGovern Institute and Broad Institute. While CRISPR-Cas9 continues to have a profound effect and huge potential for research and biomedical, and agricultural applications, the ability to move entire genes into specific target locations remained out reach.

“Genome editing with CRISPR-Cas enzymes typically involves cutting and disrupting genes, or making certain base edits,” explains Strecker, “however, inserting large pieces of DNA is still hard to accomplish.”

As a postdoctoral researcher in the lab of CRISPR pioneer Feng Zhang, Strecker led research that showed how large sequences could be inserted into a genome at a given location.

“Nature often has interesting solutions to these problems and we were fortunate to identify and characterize a remarkable CRISPR system from cyanobacteria that functions as a programmable transposase.”

Importantly, the system he discovered, called CAST, doesn’t require cellular machinery to insert DNA. This is important as it means that CAST could work in many cell types, including those that have stopped dividing such as neurons, something that is being pursued.

By finding new sources of inspiration, be it nature or art, both Rodriques and Strecker join a stellar line up of young investigators being recognized for creativity and innovation.

 

Brain region linked to altered social interactions in autism model

by Sabbi Lall |

Although psychiatric disorders can be linked to particular genes, the brain regions and mechanisms underlying particular disorders are not well-understood. Mutations or deletions of the SHANK3 gene are strongly associated with autism spectrum disorder (ASD) and a related rare disorder called Phelan-McDermid syndrome. Mice with SHANK3 mutations also display some of the traits associated with autism, including avoidance of social interactions, but the brain regions responsible for this behavior have not been identified.

A new study by neuroscientists at MIT and colleagues in China provides clues to the neural circuits underlying social deficits associated with ASD. The paper, published in Nature Neuroscience, found that structural and functional impairments in the anterior cingulate cortex (ACC) of SHANK3 mutant mice are linked to altered social interactions.

“Neurobiological mechanisms of social deficits are very complex and involve many brain regions, even in a mouse model,” explains Guoping Feng, the James W. and Patricia T. Poitras Professor at MIT and one of the senior authors of the study. “These findings add another piece of the puzzle to mapping the neural circuits responsible for this social deficit in ASD models.”

The Nature Neuroscience paper is the result of a collaboration between Feng, who is also an investigator at MIT’s McGovern Institute and a senior scientist in the Broad Institute’s Stanley Center for Psychiatric Research, and Wenting Wang and Shengxi Wu at the Fourth Military Medical University, Xi’an, China.

A number of brain regions have been implicated in social interactions, including the prefrontal cortex (PFC) and its projections to brain regions including the nucleus accumbens and habenula, but these studies failed to definitively link the PFC to altered social interactions seen in SHANK3 knockout mice.

In the new study, the authors instead focused on the ACC, a brain region noted for its role in social functions in humans and animal models. The ACC is also known to play a role in fundamental cognitive processes, including cost-benefit calculation, motivation, and decision making.

In mice lacking SHANK3, the researchers found structural and functional disruptions at the synapses, or connections, between excitatory neurons in the ACC. The researchers went on to show that the loss of SHANK3 in excitatory ACC neurons alone was enough to disrupt communication between these neurons and led to unusually reduced activity of these neurons during behavioral tasks reflecting social interaction.

Having implicated these ACC neurons in social preferences and interactions in SHANK3 knockout mice, the authors then tested whether activating these same neurons could rescue these behaviors. Using optogenetics and specfic drugs, the researchers activated the ACC neurons and found improved social behavior in the SHANK3 mutant mice.

“Next, we are planning to explore brain regions downstream of the ACC that modulate social behavior in normal mice and models of autism,” explains Wenting Wang, co-corresponding author on the study. “This will help us to better understand the neural mechanisms of social behavior, as well as social deficits in neurodevelopmental disorders.”

Previous clinical studies reported that anatomical structures in the ACC were altered and/or dysfunctional in people with ASD, an initial indication that the findings from SHANK3 mice may also hold true in these individuals.

The research was funded, in part, by the Natural Science Foundation of China. Guoping Feng was supported by NIMH grant no. MH097104, the  Poitras Center for Psychiatric Disorders Research at the McGovern Institute at MIT, and the Hock E. Tan and K. Lisa Yang Center for Autism Research at the McGovern Institute at MIT.

New CRISPR platform expands RNA editing capabilities

by Sabbi Lall |

CRISPR-based tools have revolutionized our ability to target disease-linked genetic mutations. CRISPR technology comprises a growing family of tools that can manipulate genes and their expression, including by targeting DNA with the enzymes Cas9 and Cas12 and targeting RNA with the enzyme Cas13. This collection offers different strategies for tackling mutations. Targeting disease-linked mutations in RNA, which is relatively short-lived, would avoid making permanent changes to the genome. In addition, some cell types, such as neurons, are difficult to edit using CRISPR/Cas9-mediated editing, and new strategies are needed to treat devastating diseases that affect the brain.

McGovern Institute Investigator and Broad Institute of MIT and Harvard core member Feng Zhang and his team have now developed one such strategy, called RESCUE (RNA Editing for Specific C to U Exchange), described in the journal Science.

Zhang and his team, including first co-authors Omar Abudayyeh and Jonathan Gootenberg (both now McGovern Fellows), made use of a deactivated Cas13 to guide RESCUE to targeted cytosine bases on RNA transcripts, and used a novel, evolved, programmable enzyme to convert unwanted cytosine into uridine — thereby directing a change in the RNA instructions. RESCUE builds on REPAIR, a technology developed by Zhang’s team that changes adenine bases into inosine in RNA.

RESCUE significantly expands the landscape that CRISPR tools can target to include modifiable positions in proteins, such as phosphorylation sites. Such sites act as on/off switches for protein activity and are notably found in signaling molecules and cancer-linked pathways.

“To treat the diversity of genetic changes that cause disease, we need an array of precise technologies to choose from. By developing this new enzyme and combining it with the programmability and precision of CRISPR, we were able to fill a critical gap in the toolbox,” says Zhang, the James and Patricia Poitras Professor of Neuroscience at MIT. Zhang also has appointments in MIT’s departments of Brain and Cognitive Sciences and Biological Engineering.

Expanding the reach of RNA editing to new targets

The previously developed REPAIR platform used the RNA-targeting CRISPR/Cas13 to direct the active domain of an RNA editor, ADAR2, to specific RNA transcripts where it could convert the nucleotide base adenine to inosine, or letters A to I. Zhang and colleagues took the REPAIR fusion, and evolved it in the lab until it could change cytosine to uridine, or C to U.

RESCUE can be guided to any RNA of choice, then perform a C-to-U edit through the evolved ADAR2 component of the platform. The team took the new platform into human cells, showing that they could target natural RNAs in the cell as well as 24 clinically relevant mutations in synthetic RNAs. They then further optimized RESCUE to reduce off-target editing, while minimally disrupting on-target editing.

New targets in sight

Expanded targeting by RESCUE means that sites regulating activity and function of many proteins through post-translational modifications, such as phosphorylation, glycosylation, and methylation can now be more readily targeted for editing.

A major advantage of RNA editing is its reversibility, in contrast to changes made at the DNA level, which are permanent. Thus, RESCUE could be deployed transiently in situations where a modification may be desirable temporarily, but not permanently. To demonstrate this, the team showed that in human cells, RESCUE can target specific sites in the RNA encoding β-catenin, that are known to be phosphorylated on the protein product, leading to a temporary increase in β-catenin activation and cell growth. If such a change was made permanently, it could predispose cells to uncontrolled cell growth and cancer, but by using RESCUE, transient cell growth could potentially stimulate wound healing in response to acute injuries.

The researchers also targeted a pathogenic gene variant, APOE4.  The APOE4 allele has consistently emerged as a genetic risk factor for the development of late-onset Alzheimer’s Disease. Isoform APOE4 differs from APOE2, which is not a risk factor, by just two differences (both C in APOE4 vs. U in APOE2). Zhang and colleagues introduced the risk-associated APOE4 RNA into cells, and showed that RESCUE can convert its signature C’s to an APOE2 sequence, essentially converting a risk to a non-risk variant.

To facilitate additional work that will push RESCUE toward the clinic as well as enable researchers to use RESCUE as a tool to better understand disease-causing mutations, the Zhang lab plans to share the RESCUE system broadly, as they have with previously developed CRISPR tools. The technology will be freely available for academic research through the non-profit plasmid repository Addgene. Additional information can be found on the Zhang lab’s webpage.

Support for the study was provided by The Phillips Family; J. and P. Poitras; the Poitras Center for Psychiatric Disorders Research; Hock E. Tan and K. Lisa Yang Center for Autism Research.; Robert Metcalfe; David Cheng; a NIH F30 NRSA 1F30-CA210382 to Omar Abudayyeh. F.Z. is a New York Stem Cell Foundation–Robertson Investigator. F.Z. is supported by NIH grants (1R01-HG009761, 1R01-222 MH110049, and 1DP1-HL141201); the Howard Hughes Medical Institute; the New York Stem Cell Foundation and G. Harold and Leila Mathers Foundations.

A chemical approach to imaging cells from the inside

by Karen Zusi |

A team of researchers at the McGovern Institute and Broad Institute of MIT and Harvard have developed a new technique for mapping cells. The approach, called DNA microscopy, shows how biomolecules such as DNA and RNA are organized in cells and tissues, revealing spatial and molecular information that is not easily accessible through other microscopy methods. DNA microscopy also does not require specialized equipment, enabling large numbers of samples to be processed simultaneously.

“DNA microscopy is an entirely new way of visualizing cells that captures both spatial and genetic information simultaneously from a single specimen,” says first author Joshua Weinstein, a postdoctoral associate at the Broad Institute. “It will allow us to see how genetically unique cells — those comprising the immune system, cancer, or the gut, for instance — interact with one another and give rise to complex multicellular life.”

The new technique is described in Cell. Aviv Regev, core institute member and director of the Klarman Cell Observatory at the Broad Institute and professor of biology at MIT, and Feng Zhang, core institute member of the Broad Institute, investigator at the McGovern Institute for Brain Research at MIT, and the James and Patricia Poitras Professor of Neuroscience at MIT, are co-authors. Regev and Zhang are also Howard Hughes Medical Institute Investigators.

The evolution of biological imaging

In recent decades, researchers have developed tools to collect molecular information from tissue samples, data that cannot be captured by either light or electron microscopes. However, attempts to couple this molecular information with spatial data — to see how it is naturally arranged in a sample — are often machinery-intensive, with limited scalability.

DNA microscopy takes a new approach to combining molecular information with spatial data, using DNA itself as a tool.

To visualize a tissue sample, researchers first add small synthetic DNA tags, which latch on to molecules of genetic material inside cells. The tags are then replicated, diffusing in “clouds” across cells and chemically reacting with each other, further combining and creating more unique DNA labels. The labeled biomolecules are collected, sequenced, and computationally decoded to reconstruct their relative positions and a physical image of the sample.

The interactions between these DNA tags enable researchers to calculate the locations of the different molecules — somewhat analogous to cell phone towers triangulating the locations of different cell phones in their vicinity. Because the process only requires standard lab tools, it is efficient and scalable.

In this study, the authors demonstrate the ability to molecularly map the locations of individual human cancer cells in a sample by tagging RNA molecules. DNA microscopy could be used to map any group of molecules that will interact with the synthetic DNA tags, including cellular genomes, RNA, or proteins with DNA-labeled antibodies, according to the team.

“DNA microscopy gives us microscopic information without a microscope-defined coordinate system,” says Weinstein. “We’ve used DNA in a way that’s mathematically similar to photons in light microscopy. This allows us to visualize biology as cells see it and not as the human eye does. We’re excited to use this tool in expanding our understanding of genetic and molecular complexity.”

Funding for this study was provided by the Simons Foundation, Klarman Cell Observatory, NIH (R01HG009276, 1R01- HG009761, 1R01- MH110049, and 1DP1-HL141201), New York Stem Cell Foundation, Simons Foundation, Paul G. Allen Family Foundation, Vallee Foundation, the Poitras Center for Affective Disorders Research at MIT, the Hock E. Tan and K. Lisa Yang Center for Autism Research at MIT, J. and P. Poitras, and R. Metcalfe. 

The authors have applied for a patent on this technology.

McGovern neuroscientists develop a new model for autism

Anne Trafton |

Using the genome-editing system CRISPR, researchers at MIT and in China have engineered macaque monkeys to express a gene mutation linked to autism and other neurodevelopmental disorders in humans. These monkeys show some behavioral traits and brain connectivity patterns similar to those seen in humans with these conditions.

Mouse studies of autism and other neurodevelopmental disorders have yielded drug candidates that have been tested in clinical trials, but none of them have succeeded. Many pharmaceutical companies have given up on testing such drugs because of the poor track record so far.

The new type of model, however, could help scientists to develop better treatment options for some neurodevelopmental disorders, says Guoping Feng, who is the James W. and Patricia Poitras Professor of Neuroscience, a member of MIT’s McGovern Institute for Brain Research, and one of the senior authors of the study.

“Our goal is to generate a model to help us better understand the neural biological mechanism of autism, and ultimately to discover treatment options that will be much more translatable to humans,” says Feng, who is also an institute member of the Broad Institute of MIT and Harvard and a senior scientist in the Broad’s Stanley Center for Psychiatric Research.

“We urgently need new treatment options for autism spectrum disorder, and treatments developed in mice have so far been disappointing. While the mouse research remains very important, we believe that primate genetic models will help us to develop better medicines and possibly even gene therapies for some severe forms of autism,” says Robert Desimone, the director of MIT’s McGovern Institute for Brain Research, the Doris and Don Berkey Professor of Neuroscience, and an author of the paper.

Huihui Zhou of the Shenzhen Institutes of Advanced Technology, Andy Peng Xiang of Sun Yat-Sen University, and Shihua Yang of South China Agricultural University are also senior authors of the study, which appears in the June 12 online edition of Nature. The paper’s lead authors are former MIT postdoc Yang Zhou, MIT research scientist Jitendra Sharma, Broad Institute group leader Rogier Landman, and Qiong Ke of Sun Yat-Sen University. The research team also includes Mriganka Sur, the Paul and Lilah E. Newton Professor in the Department of Brain and Cognitive Sciences and a member of MIT’s Picower Institute for Learning and Memory.

Gene variants

Scientists have identified hundreds of genetic variants associated with autism spectrum disorder, many of which individually confer only a small degree of risk. In this study, the researchers focused on one gene with a strong association, known as SHANK3. In addition to its link with autism, mutations or deletions of SHANK3 can also cause a related rare disorder called Phelan-McDermid Syndrome, whose most common characteristics include intellectual disability, impaired speech and sleep, and repetitive behaviors. The majority of these individuals are also diagnosed with autism spectrum disorder, as many of the symptoms overlap.

The protein encoded by SHANK3 is found in synapses — the junctions between brain cells that allow them to communicate with each other. It is particularly active in a part of the brain called the striatum, which is involved in motor planning, motivation, and habitual behavior. Feng and his colleagues have previously studied mice with Shank3 mutations and found that they show some of the traits associated with autism, including avoidance of social interaction and obsessive, repetitive behavior.

Although mouse studies can provide a great deal of information on the molecular underpinnings of disease, there are drawbacks to using them to study neurodevelopmental disorders, Feng says. In particular, mice lack the highly developed prefrontal cortex that is the seat of many uniquely primate traits, such as making decisions, sustaining focused attention, and interpreting social cues, which are often affected by brain disorders.

The recent development of the CRISPR genome-editing technique offered a way to engineer gene variants into macaque monkeys, which has previously been very difficult to do. CRISPR consists of a DNA-cutting enzyme called Cas9 and a short RNA sequence that guides the enzyme to a specific area of the genome. It can be used to disrupt genes or to introduce new genetic sequences at a particular location.

Members of the research team based in China, where primate reproductive technology is much more advanced than in the United States, injected the CRISPR components into fertilized macaque eggs, producing embryos that carried the Shank3 mutation.

Researchers at MIT, where much of the data was analyzed, found that the macaques with Shank3 mutations showed behavioral patterns similar to those seen in humans with the mutated gene. They tended to wake up frequently during the night, and they showed repetitive behaviors. They also engaged in fewer social interactions than other macaques.

Magnetic resonance imaging (MRI) scans also revealed similar patterns to humans with autism spectrum disorder. Neurons showed reduced functional connectivity in the striatum as well as the thalamus, which relays sensory and motor signals and is also involved in sleep regulation. Meanwhile, connectivity was strengthened in other regions, including the sensory cortex.

Michael Platt, a professor of neuroscience and psychology at the University of Pennsylvania, says the macaque models should help to overcome some of the limitations of studying neurological disorders in mice, whose behavioral symptoms and underlying neurobiology are often different from those seen in humans.

“Because the macaque model shows a much more complete recapitulation of the human behavioral phenotype, I think we should stand a much greater chance of identifying the degree to which any particular therapy, whether it’s a drug or any other intervention, addresses the core symptoms,” says Platt, who was not involved in the study.

Drug development

Within the next year, the researchers hope to begin testing treatments that may affect autism-related symptoms. They also hope to identify biomarkers, such as the distinctive functional brain connectivity patterns seen in MRI scans, that would help them to evaluate whether drug treatments are having an effect.

A similar approach could also be useful for studying other types of neurological disorders caused by well-characterized genetic mutations, such as Rett Syndrome and Fragile X Syndrome. Fragile X is the most common inherited form of intellectual disability in the world, affecting about 1 in 4,000 males and 1 in 8,000 females. Rett Syndrome, which is more rare and almost exclusively affects girls, produces severe impairments in language and motor skills and can also cause seizures and breathing problems.

“Given the limitations of mouse models, patients really need this kind of advance to bring them hope,” Feng says. “We don’t know whether this will succeed in developing treatments, but we will see in the next few years how this can help us to translate some of the findings from the lab to the clinic.”

The research was funded, in part, by the Shenzhen Overseas Innovation Team Project, the Guangdong Innovative and Entrepreneurial Research Team Program, the National Key R&D Program of China, the External Cooperation Program of the Chinese Academy of Sciences, the Patrick J. McGovern Foundation, the National Natural Science Foundation of China, the Shenzhen Science, Technology Commission, the James and Patricia Poitras Center for Psychiatric Disorders Research at the McGovern Institute at MIT, the Stanley Center for Psychiatric Research at the Broad Institute of MIT and Harvard, and the Hock E. Tan and K. Lisa Yang Center for Autism Research at the McGovern Institute at MIT. The research facilities in China where the primate work was conducted are accredited by AAALAC International, a private, nonprofit organization that promotes the humane treatment of animals in science through voluntary accreditation and assessment programs.

New gene-editing system precisely inserts large DNA sequences into cellular DNA

by Karen Zusi, Broad Institute |

A team led by researchers from Broad Institute of MIT and Harvard, and the McGovern Institute for Brain Research at MIT, has characterized and engineered a new gene-editing system that can precisely and efficiently insert large DNA sequences into a genome. The system, harnessed from cyanobacteria and called CRISPR-associated transposase (CAST), allows efficient introduction of DNA while reducing the potential error-prone steps in the process — adding key capabilities to gene-editing technology and addressing a long-sought goal for precision gene editing.

Precise insertion of DNA has the potential to treat a large swath of genetic diseases by integrating new DNA into the genome while disabling the disease-related sequence. To accomplish this in cells, researchers have typically used CRISPR enzymes to cut the genome at the site of the deleterious sequence, and then relied on the cell’s own repair machinery to stitch the old and new DNA elements together. However, this approach has many limitations.

Using Escherichia coli bacteria, the researchers have now demonstrated that CAST can be programmed to efficiently insert new DNA at a designated site, with minimal editing errors and without relying on the cell’s own repair machinery. The system holds potential for much more efficient gene insertion compared to previous technologies, according to the team.

The researchers are working to apply this editing platform in eukaryotic organisms, including plant and animal cells, for precision research and therapeutic applications.

The team molecularly characterized and harnessed CAST from two cyanobacteria, Scytonema hofmanni and Anabaena cylindrica, and additionally revealed a new way that some CRISPR systems perform in nature: not to protect bacteria from viruses, but to facilitate the spread of transposon DNA.

The work, appearing in Science, was led by first author Jonathan Strecker, a postdoctoral fellow at the Broad Institute; graduate student Alim Ladha at MIT; and senior author Feng Zhang, a core institute member at the Broad Institute, 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. Collaborators include Eugene Koonin at the National Institutes of Health.

A New Role for a CRISPR-Associated System

“One of the long-sought-after applications for molecular biology is the ability to introduce new DNA into the genome precisely, efficiently, and safely,” explains Zhang. “We have worked on many bacterial proteins in the past to harness them for editing in human cells, and we’re excited to further develop CAST and open up these new capabilities for manipulating the genome.”

To expand the gene-editing toolbox, the team turned to transposons. Transposons (sometimes called “jumping genes”) are DNA sequences with associated proteins — transposases — that allow the DNA to be cut-and-pasted into other places.

Most transposons appear to jump randomly throughout the cellular genome and out to viruses or plasmids that may also be inhabiting a cell. However, some transposon subtypes in cyanobacteria have been computationally associated with CRISPR systems, suggesting that these transposons may naturally be guided towards more-specific genetic targets. This theorized function would be a new role for CRISPR systems; most known CRISPR elements are instead part of a bacterial immune system, in which Cas enzymes and their guide RNA will target and destroy viruses or plasmids.

In this paper, the research team identified the mechanisms at work and determined that some CRISPR-associated transposases have hijacked an enzyme called Cas12k and its guide to insert DNA at specific targets, rather than just cutting the target for defensive purposes.

“We dove deeply into this system in cyanobacteria, began taking CAST apart to understand all of its components, and discovered this novel biological function,” says Strecker, a postdoctoral fellow in Zhang’s lab at the Broad Institute. “CRISPR-based tools are often DNA-cutting tools, and they’re very efficient at disrupting genes. In contrast, CAST is naturally set up to integrate genes. To our knowledge, it’s the first system of this kind that has been characterized and manipulated.”

Harnessing CAST for Genome Editing

Once all the elements and molecular requirements of the CAST system were laid bare, the team focused on programming CAST to insert DNA at desired sites in E. coli.

“We reconstituted the system in E. coli and co-opted this mechanism in a way that was useful,” says Strecker. “We reprogrammed the system to introduce new DNA, up to 10 kilobase pairs long, into specific locations in the genome.”

The team envisions basic research, agricultural, or therapeutic applications based on this platform, such as introducing new genes to replace DNA that has mutated in a harmful way — for example, in sickle cell disease. Systems developed with CAST could potentially be used to integrate a healthy version of a gene into a cell’s genome, disabling or overriding the DNA causing problems.

Alternatively, rather than inserting DNA with the purpose of fixing a deleterious version of a gene, CAST may be used to augment healthy cells with elements that are therapeutically beneficial, according to the team. For example, in immunotherapy, a researcher may want to introduce a “chimeric antigen receptor” (CAR) into a specific spot in the genome of a T cell — enabling the T cell to recognize and destroy cancer cells.

“For any situation where people want to insert DNA, CAST could be a much more attractive approach,” says Zhang. “This just underscores how diverse nature can be and how many unexpected features we have yet to find.”

Support for this study was provided in part by the Human Frontier Science Program, New York Stem Cell Foundation, Mathers Foundation, NIH (1R01-HG009761, 1R01-MH110049, and 1DP1-HL141201), Howard Hughes Medical Institute, Poitras Center for Psychiatric Disorders Research, J. and P. Poitras, and Hock E. Tan and K. Lisa Yang Center for Autism Research.

J.S. and F.Z. are co-inventors on US provisional patent application no. 62/780,658 filed by the Broad Institute, relating to CRISPR-associated transposases.

Expression plasmids are available from Addgene.