Ajay Gupta learned biology basics as a first year undergraduate on the bumpy bus ride from his small hometown to Punjab Agricultural University. Just a few hours’ ride, he made the most of his time before he returned home to help his family’s agricultural goods business.
Working extra hours in the margins of his time has become a habit for Gupta. Now a plant science first year Ph.D. student in the Bing Yang lab and Department of Plant Science and Technology Millikan Endowment recipient, Gupta starts his day around 9 a.m. and works on three plant science projects until night.
“My advisor has to sometimes push me out of the lab to go home,” Gupta said. “I have that fascination with science.”
For his Ph.D. thesis project, he studies how rice plants fight off bacterial leaf blight — a disease caused by the bacterium Xanthomonas oryzae. This disease can devastate rice fields in Asia and other areas that depend on the staple grain. His research focuses on genes that help plants resist the deadly disease.
“The second project, which is very close to my heart, … is the development of CRISPR genome editing toolkit,” Gupta said.
Gupta works to improve the gene-editing tool CRISPR-Cas9 in rice plants. Researchers currently modify chunks of DNA sequence with CRISPR, but Gupta’s goal is to modify the individual building blocks of DNA known as nucleotides. He simplifies the process to make gene editing easier for researchers with less CRISPR experience.
His third project started with self-taught skills rooted in a side passion. Gupta contributes to a global bioinformatics project investigating bacterial resistance genes in rice. By understanding the genetic make-up of plant resistance to disease, the researchers may find innovative solutions to prevent crop infections. Gupta’s input involves assembling the genome of a type of rice that is resistant to the pathogen that causes bacterial blight in rice.
“It’s a global project where everyone across the globe who uses that variety [of rice] can take advantage of that genome,” Gupta said.
Most of Gupta’s time is spent in the lab now, but Gupta started studying biology by accident. As an undergraduate he decided to major in agricultural biotechnology thinking there would be more math involved.
“It mentioned that it will have some math and some biology, and I knew nothing about biology,” Gupta said. “I said, ‘OK, it has math, I should go for it.’”
For the first year Gupta had six biology classes and one math class each semester.
“I said, ‘Oh my god, how am I going to survive,” Gupta said.
Gupta worked through basic biology courses in subjects like genetics, botany and zoology alongside his math courses. Then the math courses dropped off for the following years leaving Gupta’s schedule packed with biology classes.
“And then I started liking it,” Gupta said. “It seems more practical to me than math. I think if I would have gone to math I would have been in engineering, but I’m happy that I chose this path, and right now I’m working with plants which I love.”
Gupta’s small high school in his agricultural hometown left out many biology basics, but without the finances to attend a better school in another city Gupta was left to catch up at the university level while also helping his older brother at home.
“It was very challenging for the first year. Our financial situation was not great, so I had to support myself for my studies,” Gupta said.
Gupta returned every day to work as an accountant at his family’s small agricultural goods store to earn money for his education and to help.
“My father passed away when I was 15, and me and my brother who was 18 at that time, took care of our business without having much experience. So, I had to ride the bus four hours every day to go there and help my brother during my first year in bachelor because we could not afford at that time to hire somebody” Gupta said.
Back on their feet by his second year, Gupta continued working on weekends throughout the rest of his studies, and he only separated from the business when he left for his masters’ studies halfway around the world at South Dakota State University.
Gupta initially stayed with friends in South Dakota to soften the transition to the United States. Although difficult to live an ocean away from family, Gupta maintains relationships with his family.
“I talk with my mom almost every day. That’s a kind of an Indian or Asian thing. We are very close to our parents,” Gupta said.
Gupta started CRISPR research while working on his master’s degree at South Dakota State University. He focused on modifying wheat genes to increase yield and diversity, but on the side he also found a passion for bioinformatics.
“Most of this was self-learning, so I just found things on the internet and found out how they work and then made my own protocols and now have the pipeline,” Gupta said.
After this taste of research, Gupta was hooked. He applied to continue his studies as a Ph.D. student and asked Bing Yang, a collaborator with the lab’s wheat genetics research, for a letter of recommendation.
Instead, Yang asked Gupta to join his lab.
Gupta accepted his offer to continue working on CRISPR alongside expanding to more basic research — a change of pace from his previous work in applied agriculture.
“I want to develop a mindset where I can work on [basic science] well where there is little or no information and then I can think on my own,” Gupta said.
Gupta is the primary bioinformatics researcher in his lab and helps with data analysis for several projects alongside his bioinformatics project. While he spends most of his time hard at work on a research bench — and can often be seen walking across the Bond LSC atrium to the greenhouses late into the evening and over holidays — he also enjoys talking with lab mates.
“Sometimes, post [research] talks, we sit together and then I discuss with them if they have some problem I can try to help, and I do the same as well. If I’m having some problem I talk to them,” Gupta said.
Gupta plans to stay in academia and continue research and teaching others. He enjoys mentoring other students and also works on outreach to high school students.
“I love to do those things and talk to students, and maybe in some ways help this university as well,” Gupta said.
Gupta holds on to the inquisitive spirit that drove him to spend hours studying basic biology on the bus despite now learning in advanced research facilities.
“I love to talk with people [to] find out what they’re doing and if I can learn something out of it,” Gupta said. “Because a lot of the time our knowledge is limited and when talking to people you can get something new.”
A tickle in the throat, a stuffy nose, congestion . . . the tell-tale signs of a cold are familiar to most, and many know that with enough rest, the immune cells on standby in the human body will destroy any invaders. But what happens when plants get sick?
The Bing Yang Lab from the Bond Life Sciences Center at the University of Missouri studied this question with the Reuben Peters lab at Iowa State University by analyzing the purpose of gene clusters in rice. Along with the Zhaohu Li lab at China Agricultural University, the researchers — utilized CRISPR gene editing to determine the function of certain gene clusters in rice.
“I thought this could be something very important,” Yang said, “We can take out a huge amount of DNA to see if the gene cluster could play a role in this resistance [to disease].”
CRISPR – a gene editing tool – allows the researchers to change the DNA of the rice plants by removing sections of genetic code, known as gene clusters. By removing the cluster, scientists can see what breaks in the plant, thus identifying each cluster’s role in the plant’s health.
“These were what we termed biosynthetic gene clusters. They have unrelated genes that are close together, their physical proximity on a chromosome, and they all act in one single pathway to make a small family of natural products,” Peters said.
The scientists compared unedited rice plants with rice plants missing the gene clusters, and they determined that those without the gene clusters had decreased resistance to certain diseases. This was due to the decreased production of antimicrobial — or pathogen fighting — natural products.
The genes code for enzymes that create labdane-related diterpenoids — natural products responsible for protecting plants from infection, termed phytoalexins — a defense response.
The loss of these compounds made the plants less resistant to disease. While humans have mobile cells that can travel to pathogens through the immune system, plants rely on local chemical responses to fight off invaders.
“They’ll recognize components of the fungal cell wall, or they’ll interact with specific proteins from the fungus or the bacteria, [and] that triggers what we consider to be an immune response,” Peters said. “They have to rapidly make these compounds in what would otherwise be a general photosynthetic cell or just an epidermal cell on the surface of the leaf.”
By identifying which genes produce certain chemicals, the researchers have a greater understanding of the plant immune response, and they can manipulate the genetics of the plants to create stronger immune responses.
“We can fine tune those genes, which could increase the [protections] and make the plant more resistant to the pathogen,” Yang said.
Peters suggested since the scientists know which genes code for these proteins, they can make the proteins and create an antibiotic to prevent infections in crops and livestock.
“We do think that some of these compounds are going to be important for potential pharmaceutical activity,” Peters said. “Not necessarily the ones in rice, but related compounds, and that’s another reason we studied this.”
This research published online in New Phytologist October 16, 2021, under the title “Dissecting the labdane-related diterpenoid biosynthetic gene clusters in rice reveals directional cross-cluster phytotoxicity.”
Kulbir Sandhu’s curiosity had guided him from place to place, but it was his fascination with plant science that has stayed the same.
While Sandhu has been a postdoctoral fellow in the Bing Yang lab at Bond Life Sciences Center for the past six months, his path towards plant science began when he was 18 years old in his home country of India.
In high school, Sandhu was drawn to the biology route because of helpful and enthusiastic science teachers. He grew to like it as time went on and found that the subject came easy to him.
“I always had, you could say, a ‘scientific’ attitude,” Sandhu said. “Even when I had little understanding of the process of science, I always had an attitude that suited this field.”
Sandhu also received support and inspiration regarding science from his father, who was an engineer.
“When I was young, my dad used to help me with school homework,” Sandhu said. “His favorite subject was maths, and so he always insisted that I do well in maths and science. In this way, it became natural for me to develop an inclination for these two subjects. In India, most students interested in science choose either maths or biology streams after 10th grade. Initially, I wanted to be a doctor, so I chose biology, and it was more of a happenstance that I ended up becoming a plant ‘doctor.’”
Years later, Sandhu received his Master’s in plant breeding from the Punjab Agricultural University in Ludhiana, India in 2003, and in 2013 he received his Ph.D. at Washington University.
Sandhu first met Bing Yang four years later as a postdoctoral fellow at Iowa State University. Since they worked on a previous project together, Sandhu became a great addition to the Yang lab when he joined in November last year.
While Sandhu is working on a few projects, his main one involves using the gene-editing tool, CRISPR/CAS9, to target genes in Arabidopsis that code for reactive oxygen species. ROS helps plants with signaling, development, stress responses and other processes.
ROS is also part of the plant’s innate defense system against pathogens. Understanding how pathogens overcome this primary defense system of plants is necessary to breed better resistant crops and reduce environmental impact due to chemical control.
By causing these gene mutations, he prevents ROS from being formed in cells. That way, they can compare the mutant plant to a wild type and see the difference in basal-defense responses.
“Now this is exciting again because we are working in CRISPR in field crops as well as in basic science,” Sandhu said. “So, I get to do both things.”
First-year researcher Jack Ogilvy has been working on this project with Sandhu for the past three months as part of the Freshmen Research in Plants program.
“This is my first time … mentoring someone, and by this experience, I have realized that it is equally beneficial to me,” Sandhu said. “I mean … talking about scientific concepts helps create a deeper understanding, and both parties gain from this interaction.”
Together, the two are learning more about ROS and the Yang lab.
“He cares more about just being a mentor in terms of science,” Ogilvy said. “He also is just as interested in my personal life … We’ve formed a relationship between the two of us where it’s not just like, he tells me what to do in the lab. It’s like we are working together, essentially.”
Ogilvy appreciates Sandhu’s curiosity and advice.
“He’s always telling me to try to find the answer on my own before I go for help to gain that skill … just because it’s such an important skill to have to be somewhat self-reliant,” Ogilvy said. “But that being said, he’s always there if I get stuck or if I need help.”
Sandhu found a place for himself in the Yang lab. In a few weeks, he plans on focusing more on his own projects.
Michael Pisias came to realize that he wanted to study polyploidy while sitting in an undergraduate genetics lecture class at California State University-Sacramento (CSUS) a few years ago.
This unique phenomenon is when the cells of an organism have more than two paired sets of chromosomes, which intrigued Pisias.
“I knew I liked to figure out how living things work, especially at the smallest, genetic level,” Pisias said. “I find it fascinating that there are some creatures that are able to survive and thrive with doubled genetic information, which would be lethal to almost everything on the planet. I like to see how they’re able to do that.”
As a graduate student in the Chris Pires lab at Bond Life Sciences Center, Pisias indulges this passion studying Brassica. Modern day Brassica include many items in your grocery produce section like cabbage, cauliflower, and broccoli. These hybrids were formed by parent species millions of years ago and have evolved to contain double the genetic information.
“Essentially it’s a way to kind of generate a whole new species. These ones did it on their own, but we can do it synthetically in the lab too,” Pisias said.
Pisias focuses on the effects of the parent species’ genetic information within Brassica. He uses CRISPR to edit genomes to see how the chromosomes interact.
“It’s exciting because it’s really cutting edge,” Pisias said. “I’ve never seen a lot of the work that we’re trying to do within Brassica, a lot of this research is done in monocots like maize or wheat. It’s cool to do this in something that is further outside of what everyone else works on.”
Before Bond LSC, Pisias went to Sierra College and CSUS in his native California.
Pisias worked with local California polyploids after transferring to CSUS and learned how to be an efficient researcher through perseverance and keeping good records of his work.
“I was trying protocols repeatedly and realizing that when you’re doing research, it’s probably not going to work every time. You have to be persistent and creative,” Pisias said.
Pisias’ connections at CSUS led him to connect with Pires, and the rest is history.
“He’s bold, he’s willing to take some risks to try things that haven’t been done before,” Pires said.
At Bond LSC, Pisias has learned the value of collaborating.
“I find that a good part of my time here is spent learning from other scientists and not so much just sitting around reading things on the computer. You can learn something from everyone,” Pisias said.
He goes to neighboring labs for help and advice to advance his research.
“Everyone in the lab and in the Bond Life Sciences Center that I’ve interacted with are very friendly,” Pisias said. “They’re very inviting and I get to work with people from all over the world here. It’s been a very beneficial relationship.”
Pisias has passed it on as a mentor and teacher. He has taught Introduction to Biological Systems Lab and has helped teach a genetics course.
“I want to teach future scientists; a lot of my interest lies in mentoring other people,” Pisias said. “I currently have four undergraduate students to work with me on my research. I like to see how they can learn and grow on their own scientific journey.”
Teaching classes has helped him find undergraduate students who are enthusiastic about science and research that he ends up taking under his wing.
“He’s excited about his science and he wants to share it with people and now he has a small army of undergrads working with him,” Pires said. “He has a white board with plans for each student working on various projects. It’s a pretty serious training plan, but he’s excited for them to have a meaningful research experience.”
While he hopes to get his Ph.D., Pisias also dreams about the future of his mentees.
“I want all of them to primarily gain skills, so they can go on and be successful,” Pisias said. “In the short-term I’m hoping that they get some credit for the work that they do. It would be great if they could get the opportunity to get their names out there, potentially on some papers.”
To wind down from a long day in the lab, Pisias likes to go to the archery range to practice using his bow and arrow.
“I find it to be very relaxing, because you have to be slow and patient, which the opposite of the chaos of mentoring four undergraduate students,” Pisias laughed.
Life in the lab can be hectic, but Pisias would not want it any other way.
“I think that for us as graduate students, we’re in a really unique position. We have the privilege to mentor undergraduates who are all going to be the future scientists,” Pisias said. “I think it’s a really important responsibility that we need to take seriously.”
Patience is a virtue, at least it is for Bing Stacey.
Stacey recently completed a project that took her a total of eight years. It took her five years to develop a fast neutron mutant population and it took an additional three years to screen the population to identify a mutant that showed increase soybean seed size and then identifying the causative gene.
This gene, GmKIX8-1, and the seed size QTL, qSW17-1, can potentially be exploited for increasing yield in soybeans. Being able to alter these to increase seed size is important to improve the economic traits of soybeans such as yield and seed quality.
“The most important thing for farmers is the yield,” said Stacey, assistant professor of plant sciences at Bond Life Sciences Center. “For farmers, they plant soybeans, and the value or the profit they get is based on yield.”
There are two components of yield: seed size and seed quality. Stacey went to work finding what genes contribute to these components.
Mutants are very important for gene study because they have a new DNA sequence for genes. While it sounds like a sci-fi term, mutants are just plants manipulated using chemicals, radiations, or genetic modifications to change their characteristic traits in comparison to the norm. And the DNA changes to the GmKIX8-1 gene Stacey found is exactly the mutation she was looking for.
When she began this research, there were not many mutants for soybeans regarding seed size, so she had to develop her own mutant population using fast neutron irradiation.
After taking several years to develop this population, Stacey eventually found several mutants showing altered seed traits, including one showing increased seed size.
“So, we have the mutant, and then we utilized a fast and cost-effective way to genotype for changes in the DNA sequence of the mutant that is associated with the increased seed size. The genotyping method is called Comparative Genome Hybridization (CGH) which can be completed within one week and specifically detects missing DNA sequences in the mutant genome,” Stacey said.
Next, Stacey used CRISPR/Cas9 mutagenesis, which is a very powerful way to cut out specific DNA sequences in targeted genes in an organism. Before CRISPR, plant scientists could only create random mutations in plants. For example, using processes involving mutagenic chemicals and radiations, which meant one had to look at a very large number of mutants to screen for induced changes in the DNA sequence of specific genes.
Using fast neutron mutagenesis which creates deletions in bases, combined with CRISPR/Cas9, Stacey was able to characterize the role of the GmKIX8-1 gene in controlling seed size in soybeans.
“In a way, we got lucky because there is a QTL overlapping GmKIX8-1,” Stacey said. “And once you know the genetics behind a trait, then it is possible to define a possible mechanism of how the gene works, for example, how it makes soybean plants produce bigger seeds.” GmKIX8-1 controls the ability of cells to multiply. It also increases the size of the organs and the seeds of soybeans, which is exactly what Stacey was looking for.
An additional new and important thing Stacey found in her work was that GmKIX8-1 controls seed size, but not leaf size, in a dosage-dependent manner indicating that this gene is a major player in regulating seed size but not leaf size.
“This gene dosage effect on seed size and the identification of GmKIX8-1 as the causative gene behind a major seed size QTL are the novel aspects of this work,” Stacey said.
These novel aspects have been rewarding for Stacey.
“I’m quite happy, and it’s more fulfilling discovering something novel, something that hasn’t been reported before in other plants, instead of confirming what has been already reported,” Stacey said, “For example, based on what is already well-known in maize, or in rice, or in Arabidopsis, I can ask the question, ‘Does this gene also work in soybean?’ Answering this question is still worthwhile to me because maybe the gene will not work at all, or maybe it works in a different way, and that would also be novel, but to discover new genes or new explanations on how a gene works is more fulfilling.”
Crops resist bacterial leaf blight; ruling clears path to provide smallholder farmers with a safe, affordable option for preventing destructive disease
Columbia and St. Louis, MO, October 14, 2020 – The Healthy Crops team, with support from the Bill & Melinda Gates Foundation, have used gene editing tools to develop new varieties of disease-resistant rice that regulators in the United States and Colombia have determined are equivalent to what could be accomplished with conventional breeding. Bacterial blight can reduce rice yields by up to 70 percent, with the heaviest losses typically experienced by smallholder rice growers in low and middle-income countries. This has a profound impact on farmer productivity and economic mobility. The Healthy Crops team turned to gene editing to develop disease-resistant varieties as a way to provide farmers with a safe, affordable, effective solution.
“We first set about to understand the gene the bacteria use to make the plant vulnerable to its disease,” said Bing Yang, PhD, a researcher with the University of Missouri Bond Life Sciences Center professor, Division of Plant Sciences and member, Donald Danforth Plant Science Center in St. Louis. “We then used our CRISPR technology precisely to remove the element in the gene to avoid the pathway the pathogen takes that makes the plants susceptible to blight.”
The team used gene editing to create rice lines in elite varieties that are comparable to naturally occurring variants. These lines can resist infection by bacterial leaf blight, which leads to major losses for one of the world’s most important food crops. The rulings from the United States Department of Agriculture (USDA) and the corresponding authority in Colombia, the Instituto Colombiano Agropecuario (ICA), clear the way for field tests to select the best material for distribution to breeders in the U.S. and Colombia.
The improvements were accomplished via gene editing, which did not introduce any DNA into the plants and focused on “promoter regions” in three genes that are targeted by the causative agent of rice blight, the bacterium Xanthomonas oryzae pathovar oryzae. The research was described in an article in Nature Biotechnology in 2019.
Yang is just one member of the research consortium, headed by Humboldt Professor Wolf B. Frommer from Heinrich Heine University Düsseldorf (HHU), that has worked more than four years on this research. Six research institutions on three continents were involved including the University of Missouri, Donald Danforth Plant Science Center, University of Florida, the Alliance of Bioversity International and the International Center for Tropical Agriculture (CIAT) in Colombia, the Institut de Recherche pour le Développement (IRD) in France and the International Rice Research Institute (IRRI) in the Philippines.
In the wake of the ruling from U.S. and Colombian officials, the new blight-resistant varieties can now be used to introduce the resistance trait into many different types of rice via standard breeding strategies. Additional testing and breeding work is expected to take place in multiple locations that are favorable for growing tropical rice varieties.
“It’s exciting to use science and technology to do to help farmers protect and improve their rice production,” Yang said. “We hope to work closely with the local institutions in the next phase to introduce these into the varieties of rice small farmers use.”
The Healthy Crops Team has no commercial interest in its work. Its goal is to ensure disease- resistant rice varieties are accessible and affordable, especially for smallholder farmers who depend on rice production to support their families.
About The Donald Danforth Plant Science Center
Founded in 1998, the Donald Danforth Plant Science Center is a not-for-profit research institute with a mission to improve the human condition through plant science. Research, education and outreach aim to have impact at the nexus of food security and the environment, and position the St. Louis region as a world center for plant science. The Center’s work is funded through competitive grants from many sources, including the National Institutes of Health, U.S. Department of Energy, National Science Foundation, and the Bill & Melinda Gates Foundation. Follow us on Twitter at @DanforthCenter.
About Bond Life Sciences Center
Founded in 2004, the Christopher S. Bond Life Sciences Center was designed with teamwork in mind, fostering collaborations between scientists of diverse disciplines and backgrounds. From cancer and HIV to plant science and informatics, our researchers work together to move basic science forward and lay the groundwork for a better world. Learn more at bondlsc.missouri.edu.
up in Dhaka, the capital of Bangladesh, Sanzida Rahman longed for space to grow
a garden. She often grew plants and vegetables on small windowsills and the
roof of her home, making the most of what little space she had.
an early age, Rahman, a doctoral student in Walter Gassmann’s lab at Bond LSC,
fell in love with agriculture. She remembers visiting her family in a small
village of Bangladesh every year and helping her uncles, grandparents and
cousins on the farm.
grew up in a country that relies on agriculture for its economy. I always saw my
family doing agricultural work and I enjoyed how they did it,” Rahman said. “It
always made me feel close to nature.”
fondly recalls the sound of the rice against threshing drums and how the
threshed rice was steamed and spread out for airdrying. She also remembers how
joyful it was for her as a child, to pick ripe beans and peas from their family
chased her dream of studying science and agriculture head-on since the 10th
grade. In undergrad, she enjoyed studying entomology, crop ecology,
agroforestry, genetics and plant biology and was always open to learning new
things. After her undergraduate degree in agriculture from Khulna University Bangladesh,
Rahman received ‘VLIR-UOS fellowship’ from the Belgian government to obtain a
master’s in biology with a specialization in human ecology from Vrije
“At that point, I was open to any opportunity and I took it. The things I learned were fun and I loved the experience of studying at the heart of Europe, but I definitely wanted to get back to the agricultural track,” Rahman said.
Rahman moved to the United States to receive another master’s at North Dakota
State University, this time in plant sciences. There, she was advised by Susie
Thompson, a renowned potato breeder to expand her knowledge of plant sciences
and narrow down her interest. Now, as a plant stress biology doctoral student,
she feels she is finally settling into her field of study.
In the Gassmann lab, Rahman is working on establishing a protocol for agrobacterium-mediated lettuce transformation. Essentially, plant transformation is a scientific approach where DNA from another organism is inserted into the genome of the species of interest, in this case, lettuce. The resulting plant is then transgenic.
plants help the plant research community test hypothesis and improve the
characteristics of crops. Some traits include yield, disease resistance,
abiotic stress tolerance and nutrient abundance.
In this process of lettuce transformation, Rahman hopes to knock out the Enhanced Disease Susceptibility 1(EDS1) gene in lettuce using CRISPR-Cas9 technology in order to unlock its function in immunity.
“EDS1 is known to be a positive immune regulator for both basal and innate plant immunity, but the actual function of EDS1 and how it essentially contributes to triggering plant immunity is yet to be known. Many studies with Arabidopsis and other model plants have established the fundamentals of plant immunity, however, plants from different clades exhibit distinctly configured immune system. Our lab found that lettuce has a unique immune system compared to other model plants,” Rahman said. “It is important to study EDS1 in different plant systems so that this knowledge can be implemented upon a wide range of species for the improvement of overall plant defense,” Rahman emphasized.
her Ph.D., Rahman wants to be a post-doctoral researcher in the field of plant immunity.
Beyond that, she wants to be a professor and faculty researcher.
In her spare time, Rahman still grows vegetables on her small balcony with her husband, sings and paints. She also likes to stay close to her community.
don’t need any occasion to celebrate or do anything really. We play board
games, sometimes sing and eat our traditional foods and enjoy each other’s
company, whenever we are together. We have several community programs held every
month so we don’t feel much away from home,” Rahman said. She loves to call her
mom and dad every day, keeping a close connection with her family and friends
back home. “That’s the most important thing for me outside of my work.”
The assistant professor of comparative medicine and genetics at the University of Missouri had joined forces with a startup company developing a tool to detect early colon cancer-causing lesions. They already tried out a rat-sized model, but still needed a full-sized prototype.
Scientists in Europe had an ideal pig model for colon cancer, but importing the animals presented a problem. It would be prohibitively expensive and time consuming, and the method European scientists used to develop the pig took several years and cost a great many Euros, Amos-Landgraf said.
Those obstacles might have been enough to scuttle the project entirely, but CRISPR, a new gene-editing tool discovered in the DNA of a peculiar bacterium, has changed the equation for scientists everywhere.
So when Amos-Landgraf went to the National Swine Resource and Research Center (NSRRC) to ask about importing pigs, they told him, “‘We can just make you the model,’” Amos-Landgraf said. “‘We should be able to do a CRISPR project within a few months.’”
CRISPR is rapidly reshaping the way biologist around the world do their jobs.
At Mizzou, it’s transforming how researchers learn about viruses and mosquitoes, pigs and zebrafish, and the individual genes affecting development, sickness and health. The tool makes research more efficient, cost-effective and vastly more powerful.
Amos-Landgraf knows firsthand just how time-consuming and laborious generating an animal model was pre-CRISPR.
“What was almost a two-year process just to generate an animal now would take us a matter of months,” Amos-Landgraf said. “I think the CRISPR revolution is going to be amazing for all of science. I’m totally intrigued by everything that’s going on with this.”
Borrowing a bacterial relic
CRISPR rolls off the tongue far more readily than its unabbreviated equivalent: “clustered regularly interspaced short palindromic repeats.” The name refers to a strange pattern scientists at the University of California, Berkeley noticed in the genome of a bacterium that lives in acidic, abandoned mines: groups of palindromic bacterial DNA sequences interspersed with segments of viral DNA.
It turned out that the genetic snippets were relics of the bacteria’s prior run-ins with viral invaders, like genetic mug shots on a most-wanted list.
Viruses are tiny packages of genetic material that hijack cells, such as bacteria, in order to reproduce. And when a virus enters a bacterial cell, the host compares the virus’ genetic material to the snapshots preserved in the bacteria’s own DNA. If they match, the bacteria dispatches a bounty-hunter protein called Cas9, which tracks down the virus and slices its DNA in half at the very spot that matched the virus’ genetic fingerprint.
If an unfamiliar virus attacks and the bacterium survives, it will incorporate a segment of the invader’s DNA into its own, adding a new battle scar to its DNA and a new miscreant to the most-wanted list.
When the researchers studying the bacterial immune system figured out how it worked, they realized the process could have implications far beyond the organism’s acidic abode: It could become a powerful, inexpensive, and versatile gene-editing tool.
A ground shift
The journey to better manipulate genes has been a long one.
For decades, scientists relied on various techniques and tricks to tease out the function of genes. The most common tool is forward genetics, where a researcher starts with an interesting characteristic in an organism and then hunts for the gene that caused it. Those characteristics could be traits that occur naturally, such as genetic diseases in purebred dogs or pigmentation in corn kernels, or a scientist could induce defects — essentially altering an organism’s genome by exposing it to a bath of nasty chemicals.
Imagine that an organism is like a car, suggests Anand Chandrasekhar, a Bond Life Sciences Center biologist and professor in the division of biological sciences.
“You take a car that is running nicely and you have some kind of weird mechanic from Hell come in and mess something up — just one thing — and the car doesn’t run. Then you have to figure out why the car doesn’t run by looking carefully for where the defect is.”
Reverse genetics — unsurprisingly — starts on the other end. Researchers pick a gene of interest and try to silence or alter it. If they succeed, then they look for changes in the organism that suggest the altered gene plays a role in the observed characteristic.
This tool shaped how scientists do research and what animals they use in their labs. In fact, model organisms such as mice rose in popularity partly because of how easily reverse genetic techniques like homologous recombination work with them, said Amos-Landgraf. But this approach was time consuming, expensive and didn’t function well on other organisms.
The next step forward were Zinc-Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs). Both act like guided missiles to strike at a gene of interest, targeting a specific region of genetic material and breaking both strands of the organism’s DNA at that spot. Once the DNA is broken, the cell’s natural repair mechanism intervenes and stitches the gene back together.
However, the process is prone to errors — mutations — that can alter or silence a gene. ZFNs and TALENs reliably worked in a broader array of species.
CRISPRs represent the next advancement in this process and is far faster than previous techniques.
“Let’s say if you had 15 or 20 genes that you wanted to study: You could design a CRISPR reagent for each one of them in a couple of afternoons, whereas in the ‘olden days’ (three or four years ago) with TALENs that could have taken you months,” Chandrasekhar said. “And if you were using ZFNS… you would not even imagine doing it, because you would have been crazy.”
At MU’s NIH-funded Rat Resource and Research Center (RRRC), scientists think CRISPR will help break dependence on default model organisms. The RRRC is the only center of its kind in the US and one of two in the world, serving as a repository and distribution center for rats that model human diseases.
“We’re always preaching, use the species that’s most appropriate for the question you’re asking,” said Elizabeth Bryda, a professor of veterinary pathobiology at MU who heads the RRRC. “If you’re studying human disease, use the species that best recapitulates that disease. I think CRISPR will give people the flexibility to really work in the species they want to be working in.”
For example, the center is using CRISPR to develop rat models of human inflammatory bowel diseases, such as Crohn’s disease. “All of those barriers to making rat models are no longer issues,” Bryda said, “CRISPR is easy and finally allows us to manipulate rats in ways we haven’t before.”
That’s good news for the RRRC: “I do think we’re going to see a huge increase in the number of rat models,” Bryda said, “which would increase our inventory.”
Seeing through the zebrafish
Zebrafish are another model organism that might become even more important thanks to CRISPR.
Originally found in the rice paddies and streams of India and Myanmar, the minnow-like fish is an important model organism. They’re easy to care for, produce abundant offspring and — because their embryos are transparent — make great tools for studying development.
Chandrasekhar uses zebrafish to study cranial motor neurons, the neurons that connect to, and control, muscles in the head. His lab is especially interested in the way those cranial motor neurons are deployed during development: how the neurons know where to go and to which muscles they should link.
“CRISPR is a really big boon for research, because now even small labs can test tens of genes over a short period of time for their effect on a particular biological process,” Chandrasekhar said. “That’s how we use it: We study the process of cell migration within a nervous system, and we want to study a whole slew of associated genes.”
Researchers have identified hundreds of new and potentially important genes using advance genomics, but the old techniques of reverse genetics were too slow and tedious to keep up with the new discoveries.
“CRISPR has removed the bottleneck,” Chandrasekhar said. “We can rapidly go through and, hopefully, find new genes and new signaling pathways that might be playing a very specific role for the migration and the biological process that we study.”
But finding a new gene is just the beginning, Chandrasekhar said.
“We have one student who is testing five genes, and if even one or two of those genes turn out to be important, that will then be sufficient for the lab to continue working on them for two or three years.”
Although scientists primarily use CRISPR as Chandrasekhar does, to silence genes in model organisms, new genes can also be introduced.
Through a process called homologous directed repair, scientists select a location where they want to introduce a gene and design a CRISPR to target that region.
Daniel Davis, a PhD candidate and lab manager for Assistant Professor of Veterinary Pathobiology Catherine Hagan, is developing a technique to screen potential antidepressant drugs by leveraging CRISPR technology and the advantages of the zebrafish.
When a zebrafish is stressed, it produces a neurotoxic compound, but when the fish is calm, it produces a different compound, one that is neuro-protective. The difference depends on which key enzyme the fish produces — in a stressful situation, the fish produces more of the enzyme that leads to neurotoxicity.
Davis is using CRISPR to try to link different fluorescent proteins genes to each branch of this stress pathway: If the fish produces more of the stressful compound, it will also produce a red fluorescent protein. If the other pathway is taken, the fish will assemble green fluorescent protein.
“If you take some fish, subject them to a stressor and test a variety of potential therapeutics on them, you could visualize the fluorescent proteins to see which therapeutics are more protective,” Davis said.
Altering the host to understand the virus
Other models present special challenges. In mosquitoes, for instance, it’s hard to knock out genes from its genome using traditional methods.
“The problem is that in mosquitoes such as Aedes aegypti, ‘traditional’ knockouts never really worked, so people tried out new techniques such as ZFNs and TALENs,” said Alexander Franz, assistant professor of veterinary pathobiology at MU. But the other techniques had flaws, too: they were expensive, complicated to assemble and often posed issues of efficiency and specificity.
Franz studies arthropod-borne viruses (arboviruses), specifically dengue virus and chikungunya virus. The life cycle of an arbovirus requires its circulation between arthropods, such as mosquitoes, and vertebrate hosts, such as humans. Because vaccines exist for only a few mosquito-borne viruses — yellow fever and Japanese encephalitis, for example — people usually rely on conventional and often ineffective environmental controls to thwart disease: bed nets, the elimination of breeding areas, insecticides.
Franz is pursuing a different avenue for protection that uses genetic manipulations to interrupt the transmission cycle of a virus in the mosquito.
“If you can stop the virus from taking hold in the mosquito, you can block transmission of the virus to its vertebrate host,” he said. “But to do so, you need an effective way to manipulate the mosquito’s genome.”
This is where CRISPR comes into play. “When people started reporting using the CRISPR system for genome editing in Drosophila or zebrafish, we immediately had the idea to try it out in mosquitoes.” Working with two postdocs, Franz demonstrated for the first time that the CRISPR system was capable of disrupting genes in mosquitoes.
To do so, he started with a line of transgenic mosquitoes that had already been modified to produce red and blue fluorescent proteins in their eyes. The lab designed a CRISPR to silence the gene responsible for the blue fluorescent protein. After trying a few different methods, they found a technique that turned off the target gene when they injected the CRISPR into mosquito embryos.
Because it is a very powerful and easy-to-handle genome editing technique, CRISPR has been recently utilized and further developed by other groups studying mosquito-pathogen interactions.
Other MU researchers focus on the viral interaction with human host cells.
Marc Johnson, associate professor of molecular microbiology and immunology at the Bond Life Sciences Center, studies the way a virus puts itself together inside a host cell and fights off the cell’s defenses.
“We don’t know all the cellular genes, cellular machinery and cellular pathways that viruses are harnessing,” Johnson said. “The best way to say that a virus requires a particular gene would be to knock it out of the cell and see if the virus can still replicate.”
“CRISPR is a real ground shift in how we can do science,” he said. “Things that took 6 months to a year to make one gene before, now we can do half-a-dozen in a week.”
The technique has altered the rate at which Johnson’s research proceeds and expanded the scope of his lab’s work. “It’s allowed me to take a step back and think about the whole genome, as opposed to being totally focused on this one thing or that one thing,” Johnson said. “I’d never really taken a step back to think about the whole genome — every gene, where are they and what families. It’s changed my outlook on the cell, the way I can think about it.”
The CRISPR era
Amos-Landgraf and the researchers at NSRRC are still in the process of validating their pig model: developing primers to identify the mutation and creating the CRISPRs themselves. Once everything is ready, they’ll test out the lesion-detecting colonoscope, and if all goes well, move into human trials — far faster and more economically than would have been possible a few years ago.
But Amos-Landgraf is tantalized by the possibilities the technology offers beyond increased speed and reduced costs: “To be able to tease apart not just a single gene in a pathway, but maybe think about knocking out or altering all the genes in a pathway and looking at combinations of those pathways… You can start thinking about multiple gene knockouts, multiple gene manipulations all within the same experiment,” he said.
“And that is not only cost saving, but it becomes a really powerful tool when you want to interrogate biology. We’ve entered a new era of genetics and genomics.”