The Future of Gene Editing

In the 19th century, the Swiss military contracted for a multifunctional pocket knife for officers to carry in the field. In addition to having a short blade, the compact gadget featured a fold-out can opener and two implements vital for maintaining the standard-issue Swiss rifle: a screwdriver, essential for disassembling and reassembling the firearm for cleaning, and a reamer, used to smooth burrs in the gun’s metal barrel. Today, the Swiss Army knife comes in dozens of models, each featuring tools curated for a particular audience—gardeners, hunters, locksmiths, even oenophiles. 

The iconic tool also is an analogy for CRISPR. For 3 billion years, unicellular bacteria have deployed CRISPR—Clustered Regularly Interspaced Short Palindromic Repeats—to defend themselves against viral attacks. In 2012, scientists demonstrated that CRISPR could be reprogrammed to modify the DNA of eukaryotes. Think precision scalpel, gene silencer, gene amplifier, and—like the modern-day Swiss Army knife—an expanding inventory of additional tools. 

At the Vagelos College of Physicians and Surgeons, CRISPR has become a mainstay of discovery, with basic scientists developing new CRISPR-based tools while translational researchers put those tools to use, revealing new insights into human disease and its management. 

“There are so many variations on the core theme,” says Sam Sternberg, PhD, assistant professor of biochemistry & molecular biophysics, who—with his PhD adviser, CRISPR pioneer Jennifer Doudna—advanced the Swiss Army knife analogy in “A Crack in Creation: Gene Editing and the Unthinkable Power to Control Evolution,” a 250-page book on the discovery of CRISPR and its implications for the life sciences. 

Lessons from Bacteria

As a graduate student, Sternberg worked with Doudna to develop one of the earliest CRISPR-based tools. Since joining Columbia in 2018, Sternberg has broadened his search, looking for additional gene-editing systems found in nature’s earliest life forms and detailing how they can be deployed to advance genomic discovery in the lab. “Bacteria have been fighting off viruses for a long time and the diversification of their immune systems is a treasure trove for building new technologies,” he says. “We’re not done discovering new biology, and the more we find, the more we can leverage for tool development.” 

The work emerges from the predominantly unicellular life forms known as prokaryotes, the organisms that lack a nucleus or other dedicated organelles. With DNA floating freely throughout their cytoplasm, prokaryotes can’t afford to get sloppy about detecting and neutralizing foreign genes that could prove their undoing. Enter the innate DNA surveillance tool that serves as a protean adaptive immune system. Each time a bacterium vanquishes pathogenic DNA, it captures a few characteristic snippets—those clustered regularly interspaced short palindromic repeats—and creates an RNA copy, the genetic equivalent of a wanted poster. As the bacterium’s adaptive immune system continues surveilling the prokaryotic cytoplasm, it peruses those sheaves of wanted posters. In the event of a positive ID, it uses a CRISPR-associated protein (Cas, for short) like a precision scalpel to mount a brisk and robust defense, snipping the offending DNA to pieces and stopping the invader in its tracks. 

CRISPR has completely transformed the ways biologists study biology. It has given basic scientists a new and more powerful way of asking questions like, ‘What genes are involved in cancer becoming metastatic?’ and opened new avenues for drug development.

Eukaryotes—from fungi to humans—boast a nucleus to contain and protect DNA, making targeted gene modification a time-consuming and technically challenging enterprise. Using a combination of chemicals, electrical current, viruses, and micropipettes, technicians break through the cellular membrane and into the cell nucleus to induce breaks in the DNA—all without killing the cell. Then they rely on homologous repair, an innate quality control system that cells use to fix broken strands of DNA. (It’s a lot like patching a pair of jeans: If the patch and the hole correspond around the edges, the splice will hold.) 

Technological innovations in recent decades—gene sequencing, cell cloning, RNA interference, zinc finger nuclease technology, and transcription activator-like effector nucleases, for example—have given scientists greater control of their tinkering, allowing them to turn on or off target genes to create “knock-in” or “knockout” animals. CRISPR, however, has been transformative, allowing scientists to cut and paste strands of DNA at specific locations, all within the nucleus of living cells. First, they create a CRISPR RNA seeded with snippets of a target genetic sequence. Then they inject it, along with a Cas enzyme, into the nucleus of a eukaryote. Cas zeroes in on the location specified by the RNA and induces double-strand breaks. 

“CRISPR-Cas immune systems have completely transformed the ways biologists study biology,” says Sternberg. “It has given basic scientists a new and more powerful way of asking questions like, ‘What genes are involved in cancer becoming metastatic?’ and opened new avenues for drug development. Across campus, people are using CRISPR as a better way to design their experiments.” 

CRISPR Accelerates Science

Neuroscientist Steven Siegelbaum, PhD, has spent decades digging into the mechanisms of HCN1, a gene that serves as an electrical pacemaker within the human cortex, the part of the brain responsible for higher thought processes. In recent years, genome-wide association studies have implicated HCN1 mutations in forms of infantile and pediatric epilepsy that cannot be explained by a head injury, infection, metabolic disorder, or other clinical evidence. In some cases, seizures are so severe they lead to progressive brain dysfunction and developmental delays. 

To develop the mouse models that could reveal how those mutations wreak such havoc, Siegelbaum and associate research scientist Bina Santoro, PhD, a longtime lead investigator in the Siegelbaum lab’s HCN1 research, turned to CRISPR. “It’s very fast, it’s comparatively cheaper than the traditional way of introducing point mutations, and there are a lot of these mutations in human patients that affect different parts of the HCN1 gene,” says Santoro. “We wanted to generate not just one mouse line, but a collection of mutations in the HCN1 gene, which are also present in human patients, to see the extent to which the mice reproduce the human condition.” 

Using support from a Columbia Precision Medicine Initiative program and expertise in the Columbia transgenic mouse shared resource, Siegelbaum and Santoro have already developed four lines of mice with HCN1 mutations and seizure disorders and begun analyzing the morphology of their brains for preliminary clues about how the mutations affects brain anatomy and biochemistry. “In the best case, you save a year with CRISPR, maybe 12 to 18 months, depending on how lucky you are with the technique,” says Siegelbaum. “The general proof of principle, that these mutations are causing the seizures, will happen pretty soon.” 

Deeper understanding—about the mechanisms by which proteins altered by the mutation affect electrical activity in the brain—will take considerably longer. “By using CRISPR we know that this one mutation to HCN1 is the only one in our experimental mice,” he explains. “And if they also develop seizures, that’s strong evidence that the mutation is a cause of the disease, not just associated. That’s our goal: We want to demonstrate that it’s the HCN1 mutations in the patients that are causing the disease.” 

By simultaneously exploring multiple HCN1 variants and their role in seizures, Siegelbaum and Santoro also hope to gain insights into a basic conundrum about epilepsy, that seizure disorders take myriad forms and the drugs that can ameliorate symptoms in some patients aggravate the condition in others. “If in the mouse we can tie different mutations to different kinds of epilepsy,” says Santoro, “then we can see which mutations respond better to which drugs, or which drugs exacerbate which forms of the disease.” 

Like Siegelbaum and Santoro, Lorraine Clark, PhD, assistant medical director of the Laboratory of Personalized Genomic Medicine, mixes genome-wide association studies, basic biochemistry and functional studies, and mouse models to reveal how gene variants affect brain function. Her research focuses on such neurodegenerative diseases as Parkinson’s and essential tremor. 

Scientists already know that p.E326K, a specific variant of the glucocerebrosidase (GBA) gene, has been implicated in the severity of Gaucher disease and is one of the most common risk factors for Parkinson’s disease and dementia with Lewy bodies. Research suggests that the problem common to all three conditions has to do with how GBA encodes for the enzymes vital to the function of lysosomes, the organelles responsible for cellular digestion and waste removal. But scientists do not understand the specific mechanisms by which p.E326K disrupts enzyme production. Without that crucial insight, targeted therapies to ameliorate symptoms remain out of reach. 

To learn more about how p.E326K alters lysosomal function, Clark is combining an award from the Columbia Precision Medicine Initiative with an R03 award from the NIH to generate a mouse model that has the gene variant so she can characterize the resulting brain pathology. “CRISPR is cost-effective, convenient, and easy to use,” says Clark. “Determining the disease mechanism associated with p.E326K may open up new therapeutic targets and could have a major impact on treatment of Parkinson’s disease and dementia with Lewy bodies.” 

Creating New CRISPR Tools

Alex Chavez, MD, PhD, assistant professor of pathology & cell biology, spends roughly half of his time digging into how CRISPR works, trying to make it more effective and more efficient. He has been awarded a dozen patents; 10 of those feature CRISPR technology. This spring, Columbia filed the first patent application for his work to rapidly generate hundreds of cell lines, each with targeted mutations. “One of the tools we spend a lot of time on,” he says, “is improving how we can use CRISPR to turn genes on and off.” 

When not developing new CRISPR-based tools, Chavez uses the tools to investigate cancer and neuropathology. By activating and silencing genes implicated in such conditions as Alzheimer’s, he hopes to reveal the role of each gene in the disease process. In particular, he has homed in on the genes that help a neuronal cell tolerate proteins perturbed in Alzheimer’s and some other neurodegenerative afflictions. Healthy neuronal cells have myriad ways to protect themselves against bad proteins, including apoptosis, or programmed cell death. Chavez wants to find both the genes that buffer the effect of misfolded proteins and those that amplify their effect. “We’re looking for which levers to pull,” he says. “Nothing works in isolation. You need to know the connections to pick apart the system.” 

The same holds true of the basic biology from which CRISPR derives. To boost the understanding of how best to leverage that biology to refine existing tools and build better ones, a cadre of junior faculty convenes regularly to review the projects underway in their laboratories and troubleshoot technical challenges. They call themselves SLCC (pronounced slick) for Sternberg, Lu, Chavez, and Ciccia. “Everyone comes from different angles,” says Sternberg. Chao Lu, PhD, assistant professor of genetics & development, focuses on the epigenome—proteins that turn genes on and off. Alberto Ciccia, PhD, also assistant professor of genetics & development, investigates the mechanisms that repair DNA lesions and maintain genome integrity. Says Sternberg: “We’re each thinking about the science from a unique angle—that’s when you achieve intellectual synergy.” 

In June, Nature published the first paper from Sternberg’s lab at Columbia—a report on how a CRISPR-like system found in the bacterium Vibrio cholera can be modified to insert genetic material at a precise location, without first blasting a hole in the target DNA. Dubbed “INTEGRATE,” the new system relies on parasitic “jumping genes” known as transposons that can insert themselves into a strand of DNA, using an enzyme that works like molecular glue. “Rather than introduce DNA breaks and rely on the cell to repair the break,” says Sternberg, “INTEGRATE directly inserts a user-defined DNA sequence at a precise location in the genome, a capability that molecular biologists have sought for decades.”

CRISPR Ethics

Even as scientists develop more technically advanced CRISPR-based tools and use them to increase understanding of human disease and how to treat it, questions mount. “Scientists have been actively discussing the more technical issues,” says Sternberg, “but on the societal and ethical side, we need a lot more people present, including members of the public who will be affected— disability rights activists, disease advocacy groups, people of all stripes who just want to understand this better.” 

To promote such conversations, Sternberg has made outreach to off-campus audiences a high priority. “Part of my role here is not just to train postdocs, grad students, and undergraduates in the laboratory,” he says, “but to be involved in educating others too.” To that end, he hosts visiting high school students from the metro New York area in his laboratory and participates in a program that uses web conferencing to connect scientists with classrooms across the country. This spring, he also participated in Taste of Science, a program that invites scientists to talk about their research in local watering holes. Together with a scientist from the New York Genome Center, Sternberg discussed CRISPR at a bar in the East Village. “There were people there who have definitely never set foot in a research lab, but they’re curious,” says Sternberg. “They want to know how CRISPR can be used and want to think about how companies are going to apply this technology, what’s ethical, what’s safe, and so on.” 

Ethicist Paul S. Appelbaum, MD, was exploring the ethics of gene modification long before CRISPR came on the scene. When he launched Columbia’s Center for Research on Ethical, Legal & Social Implications of Psychiatric, Neurologic & Behavioral Genetics, CRISPR was still an obscure phenomenon observed among unicellular organisms. But in November 2018—just six years after the first paper detailing how CRISPR could be used to modify a eukaryotic cell—a Chinese scientist announced that the genomes of twin girls born earlier that month had been modified using the technology. 

While the Chinese case brought to the fore myriad technical and ethical questions about human gene editing, Appelbaum sees the central issue raised by CRISPR as this: whether particular modifications die with the individual or can be passed to the next generation. “Are we treating individual patients on one hand,” he asks, “or are we seeking interventions that will affect the next generation and subsequently future generations after that? They have very different ethical implications.” 

The current regulatory environment varies among nations, with the legality of human gene modification depending heavily on a clinician’s geography, raising the possibility that scientists with ambitions curtailed in their home countries might move to more favorably regulated environs. And, no consensus has emerged on the mechanisms that might be used to impose international standards for how CRISPR is used. “There are a variety of possibilities,” says Appelbaum. “You could have legislation that controls or proscribes use of CRISPR or other gene-editing technologies, you could have voluntary self-regulation by the research community, or rules imposed by funders, or a completely unregulated environment, in which researchers and clinicians are free to do what they want with technology that’s available to them.” 

As the technology advances, Appelbaum anticipates that society will be forced to confront profound questions about what it means to be human. “The assumption that we can identify conditions that should be extirpated from the human gene pool—assuming that were possible, which given the heterogeneous bases for many conditions is extremely unlikely—makes the question of whether it would be desirable a real one.” Consider, he suggests, the enormous creativity in mathematics demonstrated by some individuals on the autism spectrum or the cultural contributions of artists afflicted by mood disorders. “There are questions of neurodiversity,” he says, “but also the reality that the same gene, the same variant may have multiple consequences, particularly when we’re talking about complex traits.” And, he notes, it may be impossible to fully comprehend the choices we confront. “As we begin to be able to edit the gene pool it may be the case that we can’t anticipate some of the consequences of the changes we’re making.”