In the summer of 2013, John Schimenti, Biomedical Sciences and Molecular Biology and Genetics, began trying out a new, up-and-coming genetic-editing method known as CRISPR. In just a couple of months, he saw how easily the technique worked on mouse models. “The power was self evident, and it was so incredibly straightforward,” he says.
How CRISPR Works
CRISPR, which stands for “clustered regularly interspaced short palindromic repeats,” is a DNA sequence found in bacteria. The discovery of CRISPR dates back to the 1980s, when researchers at Osaka University sequenced the genomes of common bacteria. They found the repeating DNA sequences in many species but did not know its biological purpose.
Years later, scientists in the dairy industry confirmed that bacteria use CRISPR to destroy viruses. The molecular system works by incorporating “spacer” DNA sequences that match the DNA of viruses that have previously attacked the bacteria and its ancestors. When a virus attacks, RNA made from the CRISPR DNA binds to the matching viral DNA by Watson-Crick base-pairing. The RNA also binds to a DNA-cutting protein called a nuclease, and the formation of the whole complex results in the viral DNA being chopped up and destroyed. If an unknown virus attacks, the CRISPR system makes new spacer sequences to protect it against the virus in the future. With this knowledge in hand, the dairy industry identified different strains of bacteria and could see whether bacterial cultures used in products such as yogurt or cheese were immune to specific viruses.
It wasn’t until 2012, however, that researchers discovered a way to leverage the CRISPR system to slice up any DNA sequence in bacteria, viral or not. The type of CRISPR system they used involved a nuclease called Cas9. The researchers could engineer the CRISPR DNA sequence to make an RNA that matched essentially any DNA target they wanted. The RNA bound to Cas9 would do the rest, finding the matching DNA sequence and cutting it.
Since then, researchers across the globe have worked at an incredible pace to use CRISPR for further applications. Scientists have demonstrated that the CRISPR/Cas9 system works in a wide range of organisms and cells, including human cells, plants, and model organisms such as flies, worms, and mice. Many Cornell scientists, in particular, are embracing the cutting-edge technology and testing its limits.
Schimenti was among the first at Cornell to utilize CRISPR in his research. Today, he uses the technique to better understand reproduction genetics. His lab is currently looking at sequenced human DNA and identifying genes that are bioinformatically predicted to harm reproduction, specifically those that affect meiosis. Schimenti and his lab are able to use the CRISPR technique to target equivalent genes in mice as a model to understand human fertility.
CRISPR is the newest of several tools available for precision genome editing, others include zinc finger nucleases, homing endonucleases, and more recently, TAL effector nucleases (TALENs). What makes CRISPR so transformative is its simplicity and how quickly it can generate a CRISPR for a new target. “What we do with CRISPR could be done already using embryonic stem cells, but that was much more difficult,” says Schimenti. “With CRISPR, it takes no more than two to three days from designing your mutation, to having your reagent, to making the mouse. This is just unbelievable. We can make very, very precise mutations with ease and even though my lab only does this with mice, the other huge thing that this technology does is work in virtually all life forms.”
Creating Fruit Fly Models
CRISPR has especially had an impact on research using the model organism drosophila, more commonly known as the fruit fly. Prior to gene editing, researchers using drosophila made mutant flies—flies with a specific genetic change relevant to a study—somewhat at random. “You had to come up with a genetic screen or method to find that one mutation in that one fly in a large population of flies with many other mutations,” says Daniel Barbash, Molecular Biology and Genetics. “Technologies like CRISPR allow us to go in and design the mutation we want and know where it is.”
The technology’s levels of power, simplicity, and precision have completely transformed the way researchers can study genetics in fruit fly populations. Barbash’s lab works on interspecific hybrids of drosophila, for example, a cross between D. melanogaster (the “lab rat” of flies) and its sibling species D. simulans. The researchers are looking at genetic mutations to determine whether a hybrid species of fly will live. The goal is to understand how these genes work in each species.
CRISPR not only makes it possible to create genetic mutations in various species of drosophila for which genetic editing was previously difficult, it also speeds genetic editing up to the point where researchers can perform studies they hadn’t considered before.
“Before CRISPR, no one would have thought of doing an experiment looking at the effect of a single mutation in 50 to 100 different populations of drosophila because there was just no practical way to do it,” Barbash explains. “Once you see this technique, a light bulb goes off and you can do that. It will give us a much richer view of how individual genes interact with the rest of the genome in real, natural populations.”
Given the newness and ongoing development of the technology, researchers are still learning how to apply CRISPR in their labs. Barbash says that the drosophila community at Cornell has met numerous times to collaboratively implement CRISPR techniques across campus. The group shares information about different approaches, and labs are helping one another learn the best specific protocols for various use cases.
Chun Han, Molecular Biology and Genetics, is in the process of establishing CRISPR protocol in his lab. Han, too, uses drosophila and says that it has been useful to talk to other researchers on campus, such as Barbash, who are already using the tool.
Han studies dendrite morphogenesis, the process in which nerve cells establish a complex network of branches, and dendrite degeneration, the process in which these nerve cell branches degrade. Both are important in preventing inflammation and keeping tissue stable, in both flies and humans.
Unlike more traditional uses of CRISPR, in which researchers use the tool to slice up a specific DNA sequence, Han is interested in using CRISPR to modify fly genes that could play a role in neurodevelopment. For example, he would like to place a fluorescent tag on a gene to trace it within a cell. This tagging is possible by overexpressing a type of protein in the tissue, but this method can cause unwanted phenomenon, Han explains. “By using CRISPR, we modify the gene in a way that maintains the natural physiological levels,” he says. “It’s much more relevant to a normal situation.”
CRISPR versus TALENs in Plant Sciences
Adam Bogdanove, School of Integrative Plant Science, is one of the leading scientists in TALENs, a gene-editing tool that, prior to the advent of CRISPR, prompted Nature Methods to cite gene editing as “Method of the Year” in 2011. Much like CRISPR, TALENs make it possible to target any specific DNA sequence in virtually any organism. Each tool, however, has its different advantages.
“CRISPR/Cas9 has a lower barrier to entry, and it’s quick,” says Bogdanove. “We, in fact, use CRISPR in some cases to study TAL effectors and their targets in plants.” TAL effectors are the proteins that are engineered to make TALENs. In nature, TAL effectors are used by plant pathogenic bacteria to change host gene expression and cause disease.
“Before CRISPR, no one would have thought of doing an experiment looking at the effect of a single mutation in 50 to 100 different populations of drosophila, because there was just no practical way to do it,” Barbash explains. “Once you see this technique, a light bulb goes off.”
That said, Bogdanove points out that CRISPR has some disadvantages compared to TALENs. CRISPR, in its current state, has more off-target effects, meaning that it can sometimes cut genes that it is not designed to affect. In a research setting, this isn’t a big problem, since a researcher can perform control experiments to identify and account for any off-target effects. But when it comes to applications such as gene therapy and agricultural biotech, there is no room to allow for off-target effects. Under these circumstances, TALENs might be preferred.
Bogdanove says that CRISPR and TALENs are both “revolutionary technologies that Cornell is pushing the frontiers of, especially in plant sciences.” The School of Integrative Plant Science, led by Alan Collmer, is embracing these technologies to push plant biology and translational research that will bring biology into agricultural fields.
“It seems that the limiting factor going forward will not be the technology; it will be how we use it,” says Collmer. “There are a lot of people at Cornell who have deep knowledge about what kind of changes in plants would really help people, whether it’s nutrition or soil health or pathogen and insect resistance. These are all huge problems that are very real and are now addressable with plants with CRISPR and TALENs.”
Unlike previous methods of genetic editing, CRISPR and TALENs don’t leave behind foreign DNA. “It’s totally natural in the sense that we could limit ourselves, for public peace of mind, to what is naturally available within the variation of a given crop and its relatives,” says Collmer. Unlike traditional plant breeding, using these genetic-editing tools speeds up the link between discovery and translational benefit. “There is a huge effort in the plant sciences now to leverage genomics to associate genes with phenotypes,” says Collmer, which means that Cornell is in a good position to translate this knowledge into better plants and crops.
One researcher who is exploring the agricultural applications of CRISPR is Kenong Xu, School of Integrative Plant Science, Horticulture. Xu works on discovering and characterizing apple genes of horticultural and economic importance. For example, he is interested in seeing whether CRISPR can be used to modify an apple’s acidity gene. He says that, to date, he has not heard of any other scientists applying CRISPR to apples, and he thinks that the acidity gene is a good target to test whether the technology can work in the fruit.
The acidity gene is especially important to apples because, in most cases, each apple variety carries two copies of the gene. One copy is functional and the other is nonfunctional because of a mutation in the gene. When making crosses between apples, the expectation is that one-fourth of the apples will carry two malfunctioning genes. These apples have very low acidity and are therefore unacceptable in taste and have no commercial value. “You really need to remove them, and you don’t need to plant them in the orchard. Apple seedlings take several years to bear fruit, so if you have hundreds of progeny, one-quarter is a significant portion to lose,” Xu says.
“The advantage of CRISPR and TALENs compared to traditional biotechnology is that they are much more precise,” says Xu. “It’s very desirable to have a tool to manipulate important genes, including the fruit acidity gene, in their native genomic environment for better fruit.”
CRISPR at the Molecular Level
Ailong Ke, Molecular Biology and Genetics, works to better understand the CRISPR system at the molecular level. Ke specifically studies the type I CRISPR system. The CRISPR/Cas9 system currently used as a gene-editing tool is the type II CRISPR system. It’s simple and powerful because the Cas9 protein performs both the targeting and the degradation activity.
Type I CRISPR, instead, involves a large complex of proteins, called the cascade, and the Cas3 protein. The cascade targets the DNA it wants to cut, and the Cas3 protein performs the degradation activity.
“I would argue that there could be more exciting applications from the type I system,” says Ke. “The Cas9 system’s base complementarity is roughly 20 nucleotides. In the type I system it’s 30 to 35, so it’s targeting a longer stretch of DNA. Targeting and degradation happens in two steps, so it is perhaps a more regulated process and could be more specific and better controlled.”
For now, however, Ke is focused on better understanding how the type I CRISPR system works. So far, he and his lab have obtained a structure of the cascade using electron microscopy and a crystal structure of the Cas3 protein. The work on the Cas3 protein was published in Nature Structural & Molecular Biology in September 2014. Both structures capture a snapshot of how the system is working. The goal is to capture multiple snapshots to create a sort of “molecular movie” of activity.
Ke explains that the next steps are to capture more crystal structures of Cas3 and to perform single molecule experiments, meaning experiments on specific molecules that track their activity through a particular event. For example, it could demonstrate how the cascade recognizes DNA in incremental steps, how the cascade recruits Cas3, and then, how it moves away. With this information, Ke says they will be able to understand the details of the system.
A Durable Technique
What’s clear is that the CRISPR system is here to stay, and Cornell scientists are embracing the latest technology. “It’s a really powerful technology and there’s got to be more powerful applications coming out of that,” says Ke. “It will become more efficient and different from what it is now. It just takes imagination.”
“It’s constantly being tweaked. We’re talking about a technology that’s only been around for a year and half,” Schimenti says. “There are literally thousands of scientists who have jumped on this—it’s being improved all the time—and people at Cornell are part of this community.”
Schimenti is also the Director of Cornell’s Stem Cell and Transgenic Core Facility, supported by NYSTEM. The facility recently added CRISPR services, for use in mouse models only, for the wider Cornell community, speeding along the adoption of the technology. CRISPR has quickly become the most requested service, Schimenti says. He adds that requests come from researchers across a variety of projects, from basic science to clinical research done at Cornell Weill.