Research Area: Genome Engineering

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

Rationale engineering generates a compact new tool for gene therapy

by Jennifer Michalowski |

Scientists at the McGovern Institute and the Broad Institute of MIT and Harvard have reengineered a compact RNA-guided enzyme they found in bacteria into an efficient, programmable editor of human DNA. The protein they created, called NovaIscB, can be adapted to make precise changes to the genetic code, modulate the activity of specific genes, or carry out other editing tasks. Because its small size simplifies delivery to cells, NovaIscB’s developers say it is a promising candidate for developing gene therapies to treat or prevent disease.

The study was led by McGovern Institute investigator Feng Zhang, who is also the James and Patricia Poitras Professor of Neuroscience at MIT, a Howard Hughes Medical Institute investigator, and a core member of the Broad Institute. Zhang and his team reported their work today in the journal Nature Biotechnology.

Compact tools

NovaIscB is derived from a bacterial DNA cutter that belongs to a family of proteins called IscBs, which Zhang’s lab discovered in 2021. IscBs are a type of OMEGA system, the evolutionary ancestors to Cas9, which is part of the bacterial CRISPR system that Zhang and others have developed into powerful genome-editing tools. Like Cas9, IscB enzymes cut DNA at sites specified by an RNA guide. By reprogramming that guide, researchers can redirect the enzymes to target sequences of their choosing.

IscBs had caught the team’s attention not only because they share key features of CRISPR’s DNA-cutting Cas9, but also because they are a third of its size. That would be an advantage for potential gene therapies: Compact tools are easier to deliver to cells, and with a small enzyme, researchers would have more flexibility to tinker, potentially adding new functionalities without creating tools that were too bulky for clinical use.

From their initial studies of IscBs, researchers in Zhang’s lab knew that some members of the family could cut DNA targets in human cells. None of the bacterial proteins worked well enough to be deployed therapeutically, however: The team would have to modify an IscB to ensure it could edit targets in human cells efficiently without disturbing the rest of the genome.

To begin that engineering process, Soumya Kannan, a graduate student in Zhang’s lab who is now a junior fellow at the Harvard Society of Fellows, and postdoctoral fellow Shiyou Zhu first searched for an IscB that would make good starting point. They tested nearly 400 different IscB enzymes that can be found in bacteria. Ten were capable of editing DNA in human cells.

Even the most active of those would need to be enhanced to make it a useful genome editing tool. The challenge would be increasing the enzyme’s activity, but only at the sequences specified by its RNA guide. If the enzyme became more active, but indiscriminately so, it would cut DNA in unintended places. “The key is to balance the improvement of both activity and specificity at the same time,” explains Zhu.

Zhu notes that bacterial IscBs are directed to their target sequences by relatively short RNA guides, which makes it difficult to restrict the enzyme’s activity to a specific part of the genome. If an IscB could be engineered to accommodate a longer guide, it would be less likely to act on sequences beyond its intended target.

To optimize IscB for human genome editing, the team leveraged information that graduate student Han Altae-Tran, who is now a postdoctoral fellow at the University of Washington, had learned about the diversity of bacterial IscBs and how they evolved. For instance, the researchers noted that IscBs that worked in human cells included a segment they called REC, which was absent in other IscBs. They suspected the enzyme might need that segment to interact with the DNA in human cells. When they took a closer look at the region, structural modeling suggested that by slightly expanding part of the protein, REC might also enable IscBs to recognize longer RNA guides.

Based on these observations, the team experimented with swapping in parts of REC domains from different IscBs and Cas9s, evaluating how each change impacted the protein’s function. Guided by their understanding of how IscBs and Cas9s interact with both DNA and their RNA guides, the researchers made additional changes, aiming to optimize both efficiency and specificity.

In the end, they generated a protein they called NovaIscB, which was over 100 times more active in human cells than the IscB they had started with and that had demonstrated good specificity for its targets.

Kannan and Zhu constructed and screened hundreds of new IscBs before arriving at NovaIscB—and every change they made to the original protein was strategic. Their efforts were guided by their team’s knowledge of IscBs’ natural evolution as well as predictions of how each alteration would impact the protein’s structure, made using an artificial intelligence tool called AlphaFold2. Compared to traditional methods of introducing random changes into a protein and screening for their effects, this rational engineering approach greatly accelerated the team’s ability to identify a protein with the features they were looking for.

The team demonstrated that NovaIscB is a good scaffold for a variety of genome editing tools. “It biochemically functions very similarly to Cas9, and that makes it easy to port over tools that were already optimized with the Cas9 scaffold,” Kannan says. With different modifications, the researchers used NovaIscB to replace specific letters of the DNA code in human cells and to change the activity of targeted genes.

Importantly, the NovaIscB-based tools are compact enough to be easily packaged inside a single adeno-associated virus (AAV)—the vector most commonly used to safely deliver gene therapy to patients. Because they are bulkier, tools developed using Cas9 can require a more complicated delivery strategy.

Demonstrating NovaIscB’s potential for therapeutic use, Zhang’s team created a tool called OMEGAoff that adds chemical markers to DNA to dial down the activity of specific genes. They programmed OMEGAoff to repress a gene involved in cholesterol regulation, then used AAV to deliver the system to the livers of mice, leading to lasting reductions in cholesterol levels in the animals’ blood.

The team expects that NovaIscB can be used to target genome editing tools to most human genes, and look forward to seeing how other labs deploy the new technology. They also hope others will adopt their evolution-guided approach to rational protein engineering. “Nature has such diversity and its systems have different advantages and disadvantages,” Zhu says. “By learning about that natural diversity, we can make the systems we are trying to engineer better and better.”

This study was funded in part by the K. Lisa Yang and Hock E. Tan Center for Molecular Therapeutics at MIT, Broad Institute Programmable Therapeutics Gift Donors, Pershing Square Foundation, William Ackman, Neri Oxman, the Phillips family, and J. and P. Poitras.

Scientists engineer CRISPR enzymes that evade the immune system

by Allessandra DiCorato | Broad Institute |

The core components of CRISPR-based genome-editing therapies are bacterial proteins called nucleases that can stimulate unwanted immune responses in people, increasing the chances of side effects and making these therapies potentially less effective.

Researchers at the Broad Institute of MIT and Harvard and Cyrus Biotechnology have now engineered two CRISPR nucleases, Cas9 and Cas12, to mask them from the immune system. The team identified protein sequences on each nuclease that trigger the immune system and used computational modeling to design new versions that evade immune recognition. The engineered enzymes had similar gene-editing efficiency and reduced immune responses compared to standard nucleases in mice.

Appearing today in Nature Communications, the findings could help pave the way for safer, more efficient gene therapies. The study was led by Feng Zhang, a core institute member at the Broad and an Investigator at the McGovern Institute for Brain Research at MIT.

“As CRISPR therapies enter the clinic, there is a growing need to ensure that these tools are as safe as possible, and this work tackles one aspect of that challenge,” said Zhang, who is also a co-director of the K. Lisa Yang and Hock E. Tan Center for Molecular Therapeutics, the James and Patricia Poitras Professor of Neuroscience, and a professor at MIT. He is an Investigator at the Howard Hughes Medical Institute.

Rumya Raghavan, a graduate student in Zhang’s lab when the study began, and Mirco Julian Friedrich, a postdoctoral scholar in Zhang’s lab, were co-first authors on the study.

“People have known for a while that Cas9 causes an immune response, but we wanted to pinpoint which parts of the protein were being recognized by the immune system and then engineer the proteins to get rid of those parts while retaining its function,” said Raghavan.

“Our goal was to use this information to create not only a safer therapy, but one that is potentially even more effective because it is not being eliminated by the immune system before it can do its job,” added Friedrich.

In search of immune triggers

Many CRISPR-based therapies use nucleases derived from bacteria. About 80 percent of people have pre-existing immunity to these proteins through everyday exposure to these bacteria, but scientists didn’t know which parts of the nucleases the immune system recognized.

To find out, Zhang’s team used a specialized type of mass spectrometry to identify and analyze the Cas9 and Cas 12 protein fragments recognized by immune cells. For each of two nucleases — Cas9 from Streptococcus pyogenes and Cas12 from Staphylococcus aureus — they identified three short sequences, about eight amino acids long, that evoked an immune response. They then partnered with Cyrus Biotechnology, a company co-founded by University of Washington biochemist David Baker that develops structure-based computational tools to design proteins that evade the immune response. After Zhang’s team identified immunogenic sequences in Cas9 and Cas12, Cyrus used these computational approaches to design versions of the nucleases that did not include the immune-triggering sequences.

Zhang’s lab used prediction software to validate that the new nucleases were less likely to trigger immune responses. Next, the team engineered a panel of new nucleases informed by these predictions and tested the most promising candidates in human cells and in mice that were genetically modified to bear key components of the human immune system. In both cases, they found that the engineered enzymes resulted in significantly reduced immune responses compared to the original nucleases, but still cut DNA at the same efficiency.

Minimally immunogenic nucleases are just one part of safer gene therapies, Zhang’s team says. In the future, they hope their methods may also help scientists design delivery vehicles to evade the immune system.

This study was funded in part by the Poitras Center for Psychiatric Disorders Research, the K. Lisa. Yang and Hock E. Tan Center for Molecular Therapeutics in Neuroscience and the Hock E. Tan and K. Lisa Yang Center for Autism Research at MIT.

Feng Zhang awarded 2024 National Medal of Technology

by Julie Pryor |

This post is adapted from an MIT News story.

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Feng Zhang, the James and Patricia Poitras Professor of Neuroscience at MIT and an Investigator at the McGovern Institute, has won the National Medal of Technology and Innovation, the nation’s highest recognition for scientists and engineers. The prestigious award recognizes “American innovators whose vision, intellect, creativity, and determination have strengthened America’s economy and improved our quality of life.”

Zhang, who is also a professor of brain and cognitive sciences and biological engineering at MIT, a core member of the Broad Institute of MIT and Harvard, and an investigator with the Howard Hughes Medical Institute, was recognized for his work developing molecular tools, including the CRISPR genome-editing system, that have accelerated biomedical research and led to the first FDA-approved gene editing therapy.

This year, the White House awarded the National Medal of Science to 14 recipients and named nine individual awardees of the National Medal of Technology and Innovation, along with two organizations. Zhang is among four MIT faculty members who were awarded the nation’s highest honors for exemplary achievement and leadership in science and technology.

Designing molecular tools

Zhang, who earned his undergraduate degree from Harvard University in 2004, has contributed to the development of multiple molecular tools to accelerate the understanding of human disease. While a graduate student at Stanford University, from which he received his PhD in 2009, Zhang worked in the lab of Professor Karl Deisseroth. There, he worked on a protein called channelrhodopsin, which he and Deisseroth believed held potential for engineering mammalian cells to respond to light.

The resulting technique, known as optogenetics, is now used widely used in neuroscience and other fields. By engineering neurons to express light-sensitive proteins such as channelrhodopsin, researchers can either stimulate or silence the cells’ electrical impulses by shining different wavelengths of light on them. This has allowed for detailed study of the roles of specific populations of neurons in the brain, and the mapping of neural circuits that control a variety of behaviors.

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

“I had no idea what CRISPR was, but I was interested in nucleases,” Zhang told MIT News in 2016. “I went to look up CRISPR, and that’s when I realized you might be able to engineer it for use for genome editing.”

In January 2013, Zhang and members of his lab reported that they had successfully used CRISPR to edit genes in mammalian cells. The CRISPR system includes a nuclease called Cas9, which can be directed to cut a specific genetic target by RNA molecules known as guide strands.

Since then, scientists in fields from medicine to plant biology have used CRISPR to study gene function and modify faulty genes that cause disease. More recently, Zhang’s lab has devised many enhancements to the original CRISPR system, such as making the targeting more precise and preventing unintended cuts in the wrong locations. In 2023, the FDA approved Casgevy, a CRISPR gene therapy based on Zhang’s discoveries, for the treatment of sickle cell disease and beta thalassemia.

The National Medal of Technology and Innovation was established in 1980 and is administered for the White House by the U.S. Department of Commerce’s Patent and Trademark Office. The award recognizes those who have made lasting contributions to America’s competitiveness and quality of life and helped strengthen the nation’s technological workforce.

The promise of gene therapy

by Robert Desimone |

Portrait of Bob Desimone wearing a suit and tie.
McGovern Institute Director Robert Desimone. Photo: Steph Stevens

As we start 2024, I hope you can join me in celebrating a historic recent advance: the FDA approval of Casgevy, a bold new treatment for devastating sickle cell disease and the world’s first approved CRISPR gene therapy.

Developed by Vertex Pharmaceuticals and CRISPR Therapeutics, we are proud to share that this pioneering therapy licenses the CRISPR discoveries of McGovern scientist and Poitras Professor of Neuroscience Feng Zhang.

It is amazing to think that Feng’s breakthrough work adapting CRISPR-Cas9 for genome editing in eukaryotic cells was published only 11 years ago today in Science.

Incredibly, CRISPR-Cas9 rapidly transitioned from proof-of-concept experiments to an approved treatment in just over a decade.

McGovern scientists are determined to maintain the momentum!

 

Incredibly, CRISPR-Cas9 rapidly transitioned from proof-of-concept experiments to an approved treatment in just over a decade.

Our labs are creating new gene therapies that are already in clinical trials or preparing to enroll patients in trials. For instance, Feng Zhang’s team has developed therapies currently in clinical trials for lymphoblastic leukemia and beta thalassemia, while another McGovern researcher, Guoping Feng, the Poitras Professor of Brain and Cognitive Sciences at MIT, has made advancements that lay the groundwork for a new gene therapy to treat a severe form of autism spectrum disorder. It is expected to enter clinical trials later this year. Moreover, McGovern fellows Omar Abudayyeh and Jonathan Gootenberg created programmable genomic tools that are now licensed for use in monogenic liver diseases and autoimmune disorders.

These exciting innovations stem from your steadfast support of our high-risk, high-reward research. Your generosity is enabling our scientists to pursue basic research in other areas with potential therapeutic applications in the future, such as mechanisms of pain, addiction, the connections between the brain and gut, the workings of memory and attention, and the bi-directional influence of artificial intelligence on brain research. All of this fundamental research is being fueled by major new advances in technology, many of them developed here.

As we enter a new year filled with anticipation following our inaugural gene therapy, I want to express my heartfelt gratitude for your invaluable support in advancing our research programs. Your role in pushing our research to new heights is valued by all faculty, students, and researchers at the McGovern Institute. We can’t wait to share our continued progress with you.

Thank you again for partnering with us to make great scientific achievements possible.

With appreciation and best wishes,

Robert Desimone, PhD
Director, McGovern Institute
Doris and Don Berkey Professor of Neuroscience, MIT

Search algorithm reveals nearly 200 new kinds of CRISPR systems

by Allessandra DiCorato | Broad Institute |

Microbial sequence databases contain a wealth of information about enzymes and other molecules that could be adapted for biotechnology. But these databases have grown so large in recent years that they’ve become difficult to search efficiently for enzymes of interest.

Now, scientists at the Broad Institute of MIT and Harvard, the McGovern Institute for Brain Research at MIT, and the National Center for Biotechnology Information (NCBI) at the National Institutes of Health have developed a new search algorithm that has identified 188 kinds of new rare CRISPR systems in bacterial genomes, encompassing thousands of individual systems. The work appears today in Science.

The algorithm, which comes from the lab of CRISPR pioneer Feng Zhang, uses big-data clustering approaches to rapidly search massive amounts of genomic data. The team used their algorithm, called Fast Locality-Sensitive Hashing-based clustering (FLSHclust) to mine three major public databases that contain data from a wide range of unusual bacteria, including ones found in coal mines, breweries, Antarctic lakes, and dog saliva. The scientists found a surprising number and diversity of CRISPR systems, including ones that could make edits to DNA in human cells, others that can target RNA, and many with a variety of other functions.

The new systems could potentially be harnessed to edit mammalian cells with fewer off-target effects than current Cas9 systems. They could also one day be used as diagnostics or serve as molecular records of activity inside cells.

The researchers say their search highlights an unprecedented level of diversity and flexibility of CRISPR and that there are likely many more rare systems yet to be discovered as databases continue to grow.

“Biodiversity is such a treasure trove, and as we continue to sequence more genomes and metagenomic samples, there is a growing need for better tools, like FLSHclust, to search that sequence space to find the molecular gems,” said Zhang, a co-senior author on the study and a core institute member at the Broad.

Zhang is also an investigator at the McGovern Institute for Brain Research at MIT, the James and Patricia Poitras Professor of Neuroscience at MIT with joint appointments in the departments of Brain and Cognitive Sciences and Biological Engineering, and an investigator at the Howard Hughes Medical Institute. Eugene Koonin, a distinguished investigator at the NCBI, is co-senior author on the study as well.

Searching for CRISPR

CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats, is a bacterial defense system that has been engineered into many tools for genome editing and diagnostics.

To mine databases of protein and nucleic acid sequences for novel CRISPR systems, the researchers developed an algorithm based on an approach borrowed from the big data community. This technique, called locality-sensitive hashing, clusters together objects that are similar but not exactly identical. Using this approach allowed the team to probe billions of protein and DNA sequences — from the NCBI, its Whole Genome Shotgun database, and the Joint Genome Institute — in weeks, whereas previous methods that look for identical objects would have taken months. They designed their algorithm to look for genes associated with CRISPR.

“This new algorithm allows us to parse through data in a time frame that’s short enough that we can actually recover results and make biological hypotheses,” said Soumya Kannan, who is a co-first author on the study. Kannan was a graduate student in Zhang’s lab when the study began and is currently a postdoctoral researcher and Junior Fellow at Harvard University. Han Altae-Tran, a graduate student in Zhang’s lab during the study and currently a postdoctoral researcher at the University of Washington, was the study’s other co-first author.

“This is a testament to what you can do when you improve on the methods for exploration and use as much data as possible,” said Altae-Tran. “It’s really exciting to be able to improve the scale at which we search.”

New systems

In their analysis, Altae-Tran, Kannan, and their colleagues noticed that the thousands of CRISPR systems they found fell into a few existing and many new categories. They studied several of the new systems in greater detail in the lab.

They found several new variants of known Type I CRISPR systems, which use a guide RNA that is 32 base pairs long rather than the 20-nucleotide guide of Cas9. Because of their longer guide RNAs, these Type I systems could potentially be used to develop more precise gene-editing technology that is less prone to off-target editing. Zhang’s team showed that two of these systems could make short edits in the DNA of human cells. And because these Type I systems are similar in size to CRISPR-Cas9, they could likely be delivered to cells in animals or humans using the same gene-delivery technologies being used today for CRISPR.

One of the Type I systems also showed “collateral activity” — broad degradation of nucleic acids after the CRISPR protein binds its target. Scientists have used similar systems to make infectious disease diagnostics such as SHERLOCK, a tool capable of rapidly sensing a single molecule of DNA or RNA. Zhang’s team thinks the new systems could be adapted for diagnostic technologies as well.

The researchers also uncovered new mechanisms of action for some Type IV CRISPR systems, and a Type VII system that precisely targets RNA, which could potentially be used in RNA editing. Other systems could potentially be used as recording tools — a molecular document of when a gene was expressed — or as sensors of specific activity in a living cell.

Mining data

The scientists say their algorithm could aid in the search for other biochemical systems. “This search algorithm could be used by anyone who wants to work with these large databases for studying how proteins evolve or discovering new genes,” Altae-Tran said.

The researchers add that their findings illustrate not only how diverse CRISPR systems are, but also that most are rare and only found in unusual bacteria. “Some of these microbial systems were exclusively found in water from coal mines,” Kannan said. “If someone hadn’t been interested in that, we may never have seen those systems. Broadening our sampling diversity is really important to continue expanding the diversity of what we can discover.”

This work was supported by the Howard Hughes Medical Institute; K. Lisa Yang and Hock E. Tan Molecular Therapeutics Center at MIT; Broad Institute Programmable Therapeutics Gift Donors; The Pershing Square Foundation, William Ackman and Neri Oxman; James and Patricia Poitras; BT Charitable Foundation; Asness Family Foundation; Kenneth C. Griffin; the Phillips family; David Cheng; and Robert Metcalfe.

Thousands of programmable DNA-cutters found in algae, snails, and other organisms

by Jennifer Michalowski |

A diverse set of species, from snails to algae to amoebas, make programmable DNA-cutting enzymes called Fanzors—and a new study from scientists at MIT’s McGovern Institute has identified thousands of them. Fanzors are RNA-guided enzymes that can be programmed to cut DNA at specific sites, much like the bacterial enzymes that power the widely used gene-editing system known as CRISPR. The newly recognized diversity of natural Fanzor enzymes, reported September 27, 2023, in the journal Science Advances, gives scientists an extensive set of programmable enzymes that might be adapted into new tools for research or medicine.

“RNA-guided biology is what lets you make programmable tools that are really easy to use. So the more we can find, the better,” says McGovern fellow Omar Abudayyeh, who led the research with McGovern fellow Jonathan Gootenberg.

CRISPR, an ancient bacterial defense system, has made it clear how useful RNA-guided enzymes can be when they are adapted for use in the lab. CRISPR-based genome editing tools developed by McGovern investigator Feng Zhang, Abudayyeh, Gootenberg and others have changed the way scientists modify DNA, accelerating research and enabling the development of many experimental gene therapies.

Researchers have since uncovered other RNA-guide enzymes throughout the bacterial world, many with features that make them valuable in the lab. The discovery of Fanzors, whose ability to cut DNA in an RNA-guided manner was reported by Zhang’s group earlier this year, opens a new frontier of RNA-guided biology. Fanzors were the first such enzymes to be found in eukaryotic organisms—a wide group of lifeforms, including plants, animals, and fungi, defined by the membrane-bound nucleus that holds each cell’s genetic material. (Bacteria, which lack nuclei, belong to a group known as prokaryotes.)

Structural illustration of Fanzors.
Predicted structural image of Fanzors. Image: Jonathan Gootenberg and Omar Abudayyeh

“People have been searching for interesting tools in prokaryotic systems for a long time, and I think that that has been incredibly fruitful,” says Gootenberg. “Eukaryotic systems are really just a whole new kind of playground to work in.”

One hope, Abudayyeh and Gootenberg say, is that enzymes that naturally evolved in eukaryotic organisms might be better suited to function safely and efficiently in the cells of other eukaryotic organisms, including humans. Zhang’s group has shown that Fanzor enzymes can be engineered to precisely cut specific DNA sequences in human cells. In the new work, Abudayyeh and Gootenberg discovered that some Fanzors can target DNA sequences in human cells even without optimization. “The fact that they work quite efficiently in mammalian cells was really fantastic to see,” Gootenberg says.

Prior to the current study, hundreds of Fanzors had been found among eukaryotic organisms. Through an extensive search of genetic databases led by lab member Justin Lim, Gootenberg and Abudayyeh’s team has now expanded the known diversity of these enzymes by an order of magnitude.

Among the more than 3,600 Fanzors that the team found in eukaryotes and the viruses that infect them, the researchers were able to identify five different families of the enzymes. By comparing these enzymes’ precise makeup, they found evidence of a long evolutionary history.

Fanzors likely evolved from RNA-guided DNA-cutting bacterial enzymes called TnpBs. In fact, it was Fanzors’ genetic similarities to these bacterial enzymes that first caught the attention of both Zhang’s group and Gootenberg and Abudayyeh’s team.

The evolutionary connections that Gootenberg and Abudayyeh traced suggest that these bacterial predecessors of Fanzors probably entered eukaryotic cells, initiating their evolution, more than once. Some were likely transmitted by viruses, while others may have been introduced by symbiotic bacteria. The research also suggests that after they were taken up by eukaryotes, the enzymes evolved features suited to their new environment, such as a signal that allows them to enter a cell nucleus, where they have access to DNA.

Through genetic and biochemical experiments led by graduate student Kaiyi Jiang, the team determined that Fanzors have evolved a DNA-cutting active site that is distinct from that of their bacterial predecessors. This seems to allow the enzyme to cut its target sequence more precisely the ancestors of TnpB, when targeted to a sequence of DNA in a test tube, become activated and cut other sequences in the tube; Fanzors lack this promiscuous activity. When they used an RNA guide to direct the enzymes to cut specific sites in the genome of human cells, they found that certain Fanzors were able to cut these target sequences with about 10 to 20 percent efficiency.

With further research, Abudayyeh and Gootenberg hope that a variety of sophisticated genome editing tools can be developed from Fanzors. “It’s a new platform, and they have many capabilities,” says Gootenberg. “Opening up the whole eukaryotic world to these types of RNA-guided systems is going to give us a lot to work on,” Abudayyeh adds.

Nature: An unexpected source of innovative tools to study the brain

by Jennifer Michalowski |

This story originally appeared in the Fall 2023 issue of BrainScan.

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Scientist holds 3D printed phage over a natural background.
Genetic engineer Joseph Kreitz looks to the microscopic world for inspiration in Feng Zhang’s lab at the McGovern Institute. Photo: Steph Steve

In their quest to deepen their understanding of the brain, McGovern scientists take inspiration wherever it comes — and sometimes it comes from surprising sources. To develop new tools for research and innovative strategies for treating disease, they’ve drawn on proteins that organisms have been making for billions of years as well as sophisticated materials engineered for modern technology.

For McGovern investigator Feng Zhang, the natural world provides a rich source of molecules with remarkable and potentially useful functions.

Zhang is one of the pioneers of CRISPR, a programmable system for gene editing that is built from the components of a bacterial adaptive immune system. Scientists worldwide use CRISPR to modify genetic sequences in their labs, and many CRISPR-based therapies, which aim to treat disease through gene editing, are now in development. Meanwhile, Zhang and his team have continued to explore CRISPR-like systems beyond the bacteria in which they were originally discovered.

Turning to nature

This year, the search for evolutionarily related systems led Zhang’s team to a set of enzymes made by more complex organisms, including single-celled algae and hard-shell clams. Like the enzymes that power CRISPR, these newly discovered enzymes, called Fanzors, can be directed to cut DNA at specific sites by programming an RNA molecule as a guide.

Rhiannon Macrae, a scientific advisor in Zhang’s lab, says the discovery was surprising because Fanzors don’t seem to play the same role in immunity that CRISPR systems do. In fact, she says it’s not clear what Fanzors do at all. But as programmable gene editors, Fanzors might have an important advantage over current CRISPR tools — particularly for clinical applications. “Fanzor proteins are much smaller than the workhorse CRISPR tool, Cas9,” Macrae says. “This really matters when you actually want to be able to use one of these tools in a patient, because the bigger the tool, the harder it is to package and deliver to patients’ cells.”

Cryo-EM map of a Fanzor protein (gray, yellow, light blue, and pink) in complex with ωRNA (purple) and its target DNA (red). Non-target DNA strand in blue. Image: Zhang lab

Zhang’s team has thought a lot about how to get therapies to patients’ cells, and size is only one consideration. They’ve also been looking for ways to direct drugs, gene-editing tools, or other therapies to specific cells and tissues in the body. One of the lab’s leading strategies comes from another unexpected natural source: a microscopic syringe produced by certain insect-infecting bacteria.

In their search for an efficient system for targeted drug delivery, Zhang and graduate student Joseph Kreitz first considered the injection systems of bacteria-infecting viruses: needle-like structures that pierce the outer membrane of their host to deliver their own genetic material. But these viral injection systems can’t easily be freed from the rest of the virus.

Then Zhang learned that some bacteria have injection systems of their own, which they release inside their hosts after packing them with toxins. They reengineered the bacterial syringe, devising a delivery system that works on human cells. Their current system can be programmed to inject proteins — including those used for gene editing — directly into specified cell types. With further development, Zhang hopes it will work with other types of therapies, as well.

Magnetic imaging

In McGovern Associate Investigator Alan Jasanoff’s lab, researchers are designing sensors that can track the activity of specific neurons or molecules in the brain, using magnetic resonance imaging (MRI) or related forms of non-invasive imaging. These tools are essential for understanding how the brain’s cells and circuits work together to process information. “We want to give MRI a suite of metaphorical colors: sensitivities that enable us to dissect the different kinds of mechanistically significant contributors to neural activity,” he explains.

Jasanoff can tick off a list of molecules with notable roles in biology and industry that his lab has repurposed to glean more information from brain imaging. These include manganese — a metal once used to tint ancient glass; nitric oxide synthase — the enzyme that causes blushing; and iron oxide nanoparticles — tiny magnets that enable compact data storage inside computers. But Jasanoff says none of these should be considered out of place in the imaging world. “Most are pretty logical choices,” he says. “They all do different things and we use them in pretty different ways, but they are either magnetic or interact with magnetic molecules to serve our purposes for brain imaging.”

Close-up picture of manganese metal
Manganese, a metal that interacts weakly with magnetic fields, is a key component in new MRI sensors being developed in Alan Jasanoff’s lab at the McGovern Institute.

The enzyme nitric oxide synthase, for example, plays an important role in most functional MRI scans. The enzyme produces nitric oxide, which causes blood vessels to expand. This can bring a blush to the cheeks, but in the brain, it increases blood flow to bring more oxygen to busy neurons. MRI can detect this change because it is sensitive to the magnetic properties of blood.

By using blood flow as a proxy for neural activity, functional MRI scans light up active regions of the brain, but they can’t pinpoint the activity of specific cells. So Jasanoff and his team devised a more informative MRI sensor by reengineering nitric oxide synthase. Their modified enzyme, which they call NOSTIC, can be introduced into a select group of cells, where it will produce nitric oxide in response to neural activity — triggering increased blood flow and strengthening the local MRI signal. Researchers can deliver it to specific kinds of brain cells, or they can deliver it exclusively to neurons that communicate directly with one another. Then they can watch for an elevated MRI signal when those cells fire. This lets them see how information flows through the brain and tie specific cells to particular tasks.

Miranda Dawson, a graduate student in Jasanoff’s lab, is using NOSTIC to study the brain circuits that fuel addiction. She’s interested in the involvement of a brain region called the insula, which may mediate the physical sensations that people with addiction experience during drug cravings or withdrawal. With NOSTIC, Dawson can follow how the insula communicates to other parts of the brain as a rat experiences these MITstages of addiction. “We give our sensor to the insula, and then it projects to anatomically connected brain regions,” she explains. “So we’re able to delineate what circuits are being activated at different points in the addiction cycle.”

Scientist with folded arms next to a picture of a brain
Miranda Dawson uses her lab’s novel MRI sensor, NOSTIC, to illuminate the brain circuits involved in fentanyl craving and withdrawal. Photo: Steph Stevens; MRI scan: Nan Li, Souparno Ghosh, Jasanoff lab

Mining biodiversity

McGovern investigators know that good ideas and useful tools can come from anywhere. Sometimes, the key to harnessing those tools is simply recognizing their potential. But there are also opportunities for a more deliberate approach to finding them.

McGovern Investigator Ed Boyden is leading a program that aims to accelerate the discovery of valuable natural products. Called the Biodiversity Network (BioNet), the project is collecting biospecimens from around the world and systematically analyzing them, looking for molecular tools that could be applied to major challenges in science and medicine, from brain research to organ preservation. “The idea behind BioNet,” Boyden explains, “is rather than wait for chance to give us these discoveries, can we go look for them on purpose?”

Researchers uncover new CRISPR-like system in animals that can edit the human genome

by Leah Eisenstadt |

A team of researchers led by Feng Zhang at the McGovern Institute and the Broad Institute of MIT and Harvard has uncovered the first programmable RNA-guided system in eukaryotes — organisms that include fungi, plants, and animals.

In a study in Nature, the team describes how the system is based on a protein called Fanzor. They showed that Fanzor proteins use RNA as a guide to target DNA precisely, and that Fanzors can be reprogrammed to edit the genome of human cells. The compact Fanzor systems have the potential to be more easily delivered to cells and tissues as therapeutics than CRISPR/Cas systems, and further refinements to improve their targeting efficiency could make them a valuable new technology for human genome editing.

CRISPR/Cas was first discovered in prokaryotes (bacteria and other single-cell organisms that lack nuclei) and scientists including Zhang’s lab have long wondered whether similar systems exist in eukaryotes. The new study demonstrates that RNA-guided DNA-cutting mechanisms are present across all kingdoms of life.

“This new system is another way to make precise changes in human cells, complementing the genome editing tools we already have.” — Feng Zhang

“CRISPR-based systems are widely used and powerful because they can be easily reprogrammed to target different sites in the genome,” said Zhang, senior author on the study and a core institute member at the Broad, an investigator at MIT’s McGovern Institute, the James and Patricia Poitras Professor of Neuroscience at MIT, and a Howard Hughes Medical Institute investigator. “This new system is another way to make precise changes in human cells, complementing the genome editing tools we already have.”

Searching the domains of life

A major aim of the Zhang lab is to develop genetic medicines using systems that can modulate human cells by targeting specific genes and processes. “A number of years ago, we started to ask, ‘What is there beyond CRISPR, and are there other RNA-programmable systems out there in nature?’” said Zhang.

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

Two years ago, Zhang lab members discovered a class of RNA-programmable systems in prokaryotes called OMEGAs, which are often linked with transposable elements, or “jumping genes”, in bacterial genomes and likely gave rise to CRISPR/Cas systems. That work also highlighted similarities between prokaryotic OMEGA systems and Fanzor proteins in eukaryotes, suggesting that the Fanzor enzymes might also use an RNA-guided mechanism to target and cut DNA.

In the new study, the researchers continued their study of RNA-guided systems by isolating Fanzors from fungi, algae, and amoeba species, in addition to a clam known as the Northern Quahog. Co-first author Makoto Saito of the Zhang lab led the biochemical characterization of the Fanzor proteins, showing that they are DNA-cutting endonuclease enzymes that use nearby non-coding RNAs known as ωRNAs to target particular sites in the genome. It is the first time this mechanism has been found in eukaryotes, such as animals.

Unlike CRISPR proteins, Fanzor enzymes are encoded in the eukaryotic genome within transposable elements and the team’s phylogenetic analysis suggests that the Fanzor genes have migrated from bacteria to eukaryotes through so-called horizontal gene transfer.

“These OMEGA systems are more ancestral to CRISPR and they are among the most abundant proteins on the planet, so it makes sense that they have been able to hop back and forth between prokaryotes and eukaryotes,” said Saito.

To explore Fanzor’s potential as a genome editing tool, the researchers demonstrated that it can generate insertions and deletions at targeted genome sites within human cells. The researchers found the Fanzor system to initially be less efficient at snipping DNA than CRISPR/Cas systems, but by systematic engineering, they introduced a combination of mutations into the protein that increased its activity 10-fold. Additionally, unlike some CRISPR systems and the OMEGA protein TnpB, the team found that a fungal-derived Fanzor protein did not exhibit “collateral activity,” where an RNA-guided enzyme cleaves its DNA target as well as degrading nearby DNA or RNA. The results suggest that Fanzors could potentially be developed as efficient genome editors.

Co-first author Peiyu Xu led an effort to analyze the molecular structure of the Fanzor/ωRNA complex and illustrate how it latches onto DNA to cut it. Fanzor shares structural similarities with its prokaryotic counterpart CRISPR-Cas12 protein, but the interaction between the ωRNA and the catalytic domains of Fanzor is more extensive, suggesting that the ωRNA might play a role in the catalytic reactions. “We are excited about these structural insights for helping us further engineer and optimize Fanzor for improved efficiency and precision as a genome editor,” said Xu.

Like CRISPR-based systems, the Fanzor system can be easily reprogrammed to target specific genome sites, and Zhang said it could one day be developed into a powerful new genome editing technology for research and therapeutic applications. The abundance of RNA-guided endonucleases like Fanzors further expands the number of OMEGA systems known across kingdoms of life and suggests that there are more yet to be found.

“Nature is amazing. There’s so much diversity,” said Zhang. “There are probably more RNA-programmable systems out there, and we’re continuing to explore and will hopefully discover more.”

The paper’s other authors include Guilhem Faure, Samantha Maguire, Soumya Kannan, Han Altae-Tran, Sam Vo, AnAn Desimone, and Rhiannon Macrae.

Support for this work was provided by the Howard Hughes Medical Institute; Poitras Center for Psychiatric Disorders Research at MIT; K. Lisa Yang and Hock E. Tan Molecular Therapeutics Center at MIT; Broad Institute Programmable Therapeutics Gift Donors; The Pershing Square Foundation, William Ackman, and Neri Oxman; James and Patricia Poitras; BT Charitable Foundation; Asness Family Foundation; Kenneth C. Griffin; the Phillips family; David Cheng; Robert Metcalfe; and Hugo Shong.

 

Season’s Greetings from the McGovern Institute

by McGovern Institute |

This year’s holiday video (shown above) was inspired by Ev Fedorenko’s July 2022 Nature Neuroscience paper, which found similar patterns of brain activation and language selectivity across speakers of 45 different languages.

Universal language network

Ev Fedorenko uses the widely translated book “Alice in Wonderland” to test brain responses to different languages. Photo: Caitlin Cunningham

Over several decades, neuroscientists have created a well-defined map of the brain’s “language network,” or the regions of the brain that are specialized for processing language. Found primarily in the left hemisphere, this network includes regions within Broca’s area, as well as in other parts of the frontal and temporal lobes. Although roughly 7,000 languages are currently spoken and signed across the globe, the vast majority of those mapping studies have been done in English speakers as they listened to or read English texts.

To truly understand the cognitive and neural mechanisms that allow us to learn and process such diverse languages, Fedorenko and her team scanned the brains of speakers of 45 different languages while they listened to Alice in Wonderland in their native language. The results show that the speakers’ language networks appear to be essentially the same as those of native English speakers — which suggests that the location and key properties of the language network appear to be universal.

The many languages of McGovern

English may be the primary language used by McGovern researchers, but more than 35 other languages are spoken by scientists and engineers at the McGovern Institute. Our holiday video features 30 of these researchers saying Happy New Year in their native (or learned) language. Below is the complete list of languages included in our video. Expand each accordion to learn more about the speaker of that particular language and the meaning behind their new year’s greeting.

New CRISPR-based tool inserts large DNA sequences at desired sites in cells

Anne Trafton |

Building on the CRISPR gene-editing system, MIT researchers have designed a new tool that can snip out faulty genes and replace them with new ones, in a safer and more efficient way.

Using this system, the researchers showed that they could deliver genes as long as 36,000 DNA base pairs to several types of human cells, as well as to liver cells in mice. The new technique, known as PASTE, could hold promise for treating diseases that are caused by defective genes with a large number of mutations, such as cystic fibrosis.

“It’s a new genetic way of potentially targeting these really hard to treat diseases,” says Omar Abudayyeh, a McGovern Fellow at MIT’s McGovern Institute for Brain Research. “We wanted to work toward what gene therapy was supposed to do at its original inception, which is to replace genes, not just correct individual mutations.”

The new tool combines the precise targeting of CRISPR-Cas9, a set of molecules originally derived from bacterial defense systems, with enzymes called integrases, which viruses use to insert their own genetic material into a bacterial genome.

“Just like CRISPR, these integrases come from the ongoing battle between bacteria and the viruses that infect them,” says Jonathan Gootenberg, also a McGovern Fellow. “It speaks to how we can keep finding an abundance of interesting and useful new tools from these natural systems.”

Gootenberg and Abudayyeh are the senior authors of the new study, which appears today in Nature Biotechnology. The lead authors of the study are MIT technical associates Matthew Yarnall and Rohan Krajeski, former MIT graduate student Eleonora Ioannidi, and MIT graduate student Cian Schmitt-Ulms.

DNA insertion

The CRISPR-Cas9 gene editing system consists of a DNA-cutting enzyme called Cas9 and a short RNA strand that guides the enzyme to a specific area of the genome, directing Cas9 where to make its cut. When Cas9 and the guide RNA targeting a disease gene are delivered into cells, a specific cut is made in the genome, and the cells’ DNA repair processes glue the cut back together, often deleting a small portion of the genome.

If a DNA template is also delivered, the cells can incorporate a corrected copy into their genomes during the repair process. However, this process requires cells to make double-stranded breaks in their DNA, which can cause chromosomal deletions or rearrangements that are harmful to cells. Another limitation is that it only works in cells that are dividing, as nondividing cells don’t have active DNA repair processes.

The MIT team wanted to develop a tool that could cut out a defective gene and replace it with a new one without inducing any double-stranded DNA breaks. To achieve this goal, they turned to a family of enzymes called integrases, which viruses called bacteriophages use to insert themselves into bacterial genomes.

For this study, the researchers focused on serine integrases, which can insert huge chunks of DNA, as large as 50,000 base pairs. These enzymes target specific genome sequences known as attachment sites, which function as “landing pads.” When they find the correct landing pad in the host genome, they bind to it and integrate their DNA payload.

In past work, scientists have found it challenging to develop these enzymes for human therapy because the landing pads are very specific, and it’s difficult to reprogram integrases to target other sites. The MIT team realized that combining these enzymes with a CRISPR-Cas9 system that inserts the correct landing site would enable easy reprogramming of the powerful insertion system.

The new tool, PASTE (Programmable Addition via Site-specific Targeting Elements), includes a Cas9 enzyme that cuts at a specific genomic site, guided by a strand of RNA that binds to that site. This allows them to target any site in the genome for insertion of the landing site, which contains 46 DNA base pairs. This insertion can be done without introducing any double-stranded breaks by adding one DNA strand first via a fused reverse transcriptase, then its complementary strand.

Once the landing site is incorporated, the integrase can come along and insert its much larger DNA payload into the genome at that site.

“We think that this is a large step toward achieving the dream of programmable insertion of DNA,” Gootenberg says. “It’s a technique that can be easily tailored both to the site that we want to integrate as well as the cargo.”

Gene replacement

In this study, the researchers showed that they could use PASTE to insert genes into several types of human cells, including liver cells, T cells, and lymphoblasts (immature white blood cells). They tested the delivery system with 13 different payload genes, including some that could be therapeutically useful, and were able to insert them into nine different locations in the genome.

In these cells, the researchers were able to insert genes with a success rate ranging from 5 to 60 percent. This approach also yielded very few unwanted “indels” (insertions or deletions) at the sites of gene integration.

“We see very few indels, and because we’re not making double-stranded breaks, you don’t have to worry about chromosomal rearrangements or large-scale chromosome arm deletions,” Abudayyeh says.

The researchers also demonstrated that they could insert genes in “humanized” livers in mice. Livers in these mice consist of about 70 percent human hepatocytes, and PASTE successfully integrated new genes into about 2.5 percent of these cells.

The DNA sequences that the researchers inserted in this study were up to 36,000 base pairs long, but they believe even longer sequences could also be used. A human gene can range from a few hundred to more than 2 million base pairs, although for therapeutic purposes only the coding sequence of the protein needs to be used, drastically reducing the size of the DNA segment that needs to be inserted into the genome.

“The ability to site-specifically make large genomic integrations is of huge value to both basic science and biotechnology studies. This toolset will, I anticipate, be very enabling for the research community,” says Prashant Mali, a professor of bioengineering at the University of California at San Diego, who was not involved in the study.

The researchers are now further exploring the possibility of using this tool as a possible way to replace the defective cystic fibrosis gene. This technique could also be useful for treating blood diseases caused by faulty genes, such as hemophilia and G6PD deficiency, or Huntington’s disease, a neurological disorder caused by a defective gene that has too many gene repeats.

The researchers have also made their genetic constructs available online for other scientists to use.

“One of the fantastic things about engineering these molecular technologies is that people can build on them, develop and apply them in ways that maybe we didn’t think of or hadn’t considered,” Gootenberg says. “It’s really great to be part of that emerging community.”

The research was funded by a Swiss National Science Foundation Postdoc Mobility Fellowship, the U.S. National Institutes of Health, the McGovern Institute Neurotechnology Program, the K. Lisa Yang and Hock E. Tan Center for Molecular Therapeutics in Neuroscience, the G. Harold and Leila Y. Mathers Charitable Foundation, the MIT John W. Jarve Seed Fund for Science Innovation, Impetus Grants, a Cystic Fibrosis Foundation Pioneer Grant, Google Ventures, Fast Grants, the Harvey Family Foundation, and the McGovern Institute.