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CRISPR

Stem cell growth, required for kernel development, is controlled in corn by a set of genes called CLEs. But how these genes change the corn is complicated. Using CRISPR genome editing, CSHL researchers found they could change kernel yield and ear size by fine-tuning the activity of one of the CLE genes, ZmCLE7. In the image: an unmodified corn cob with normal ZmCLE7 gene activity (1) is packed with regular rows of kernels. Shutting off ZmCLE7 (2) shortened the cob, disrupted row patterns, and lowered kernel yield. However, decreasing the same gene’s activity (3) led to an increase in kernel yield, while increasing the gene’s activity (4) decreased the kernel yield. Jackson Lab/CSHL 2021

By Daniel Dunaief

The current signal works, but not as well as it might. No signal makes everything worse. Something in the middle, with a weak signal, is just right.

By using the gene-editing tool CRISPR, Cold Spring Harbor Laboratory Professor Dave Jackson has fine-tuned a developmental signal for maize, or corn, producing ears that have 15 to 26 percent more kernels. 

Dave Jackson. Photo from CSHL

Working with postdoctoral fellow Lei Liu in his lab, and Madelaine Bartlett, who is an Associate Professor at the University of Massachusetts Amherst, Jackson and his collaborators published their work earlier this week in the prestigious journal Nature Plants.

Jackson calls the ideal weakening of the CLE7 gene in the maize genome the “Goldilocks spot.” He also created a null allele (a nonfunctional variant of a gene caused by a genetic mutation) of a newly identified, partially redundant compensating CLE gene.

Indeed, the CLE7 gene is involved in a process that slows the growth of stem cells, which, in development, are cells that can become any type of cell. Jackson also mutated another CLE gene, CLE1E5.

Several members of the plant community praised the work, suggesting that it could lead to important advances with corn and other crops and might provide the kind of agricultural and technological tools that, down the road, reduce food shortages, particularly in developing nations.

“This paper provides the first example of using CRISPR to alter promoters in cereal crops,” Cristobal Uauy, Professor and Group Leader at the John Innes Centre in the United Kingdom, explained in an email. “The research is really fascinating and will be very impactful.”

While using CRISPR (whose co-creators won the Nobel Prize in Chemistry in October) has worked with tomatoes, the fact that it is possible and successful in cereal “means that it opens a new approach for the crops that provide over 60% of the world’s calories,” Uauy continued.

Uauy said he is following a similar approach in wheat, although for different target genes.

Recognizing the need to provide a subtle tweaking of the genes involved in the growth of corn that enabled this result, Uauy explained that the variation in these crops does not come from an on/off switch or a black and white trait, but rather from a gradient.

In Jackson’s research, turning off the CLE7 gene reduced the size of the cob and the overall amount of corn. Similarly, increasing the activity of that gene also reduced the yield. By lowering the gene’s activity, Jackson and his colleagues generated more kernels that were less rounded, narrower and deeper.

Uauy said that the plant genetics community will likely be intrigued by the methods, the biology uncovered and the possibility to use this approach to improve yield in cereals.

“I expect many researchers and breeders will be excited to read this paper,” he wrote.

In potentially extending this approach to other desirable characteristics, Uauy cautioned that multiple genes control traits such as drought, flood or disease resistance, which would mean that changes in the promoter of a few genes would likely improve these other traits.

“This approach will definitely have a huge role to play going forward, but it is important to state that some traits will still remain difficult to improve,” Uauy explained.

Jackson believes gene editing has considerable agricultural potential.

“The prospect of using CRISPR to improve agriculture will be a revolution,” Jackson said.

Other scientists recognized the benefits of fine-tuning gene expression.

“The most used type/ thought of mutation is deletion and therefore applied for gene knockout,” Kate Creasey Krainer, president and founder of Grow More Foundation, explained in an email. “Gene modulation is not what you expect.”

While Jackson said he was pleased with the results this time, he plans to continue to refine this technique, looking for smaller regions in the promoters of this gene as well as in other genes.

“The approach we used so far is a little like a hammer,” Jackson said. “We hope to go in with more of a scalpel to mutate specific regions of the promoters.”

Creasey Krainer, whose foundation hopes to develop capacity-building scientific resources in developing countries, believes this approach could save decades in creating viable crops to enhance food yield.

She wrote that this is “amazing and could be the green revolution for orphan staple crops.”

In the United States, the Food and Drug Administration is currently debating whether to classify food as a genetically modified organism, or GMO, if a food producer used CRISPR to alter one or more of its ingredients, rather than using genes from other species to enhance a particular trait.

To be sure, the corn Jackson used as a part of his research isn’t the same line as the elite breeding stock that the major agricultural businesses use to produce food and feedstock. In fact, the varieties they used were a part of breeding programs 20 or more years ago. It’s unclear what effect, if any, such gene editing changes might have on those crops, which companies have maximized for yield.

Nonetheless, as a proof of concept, the research Jackson’s team conducted will open the door to additional scientific efforts and, down the road, to agricultural opportunities.

“There will undoubtedly be equivalent regions which can be engineered in a whole set of crops,” Uauy wrote. “We are pursuing other genes using this methodology and are very excited by the prospect it holds to improve crop yields across diverse environments.”

From left, Zachary Lippman and Dave Jackson, professors at CSHL who are working on ways to alter promoter regions of genes to control traits in tomato and corn. Photo by Ullas Pedmale

By Daniel Dunaief

He works with tomatoes, but what he’s discovered could have applications to food and fuel crops, including corn, rice and wheat.

Using the latest gene editing technique called CRISPR, Zachary Lippman, a professor at Cold Spring Harbor Laboratory, developed ways to fine-tune traits for fruit size, branching architecture and plant shape. Called quantitative variation, these genetic changes act as a dimmer switch, potentially increasing or decreasing specific traits. This could help meet specific agricultural needs. Looking at the so-called promoter region of genes, Lippman was able to “use those genes as proof of principal” for a technique that may enable the fine-tuning of several traits.

For decades, plant breeders have been looking for naturally occurring mutations that allow them to breed those desirable traits, such as a larger fruit on a tomato or more branches on a plant. In some cases, genetic mutations have occurred naturally, altering the cell’s directions. At other times, breeders have sought ways to encourage mutations by treating their seeds with a specific mutagenic agent, like a chemical.

In an article in the journal Cell, Lippman said the results reflect a road map that other researchers or agricultural companies can use to create desirable traits. This article provides a way to “create a new, raw material for breeders to have access to tools they never had before,” he said. Lippman has taken a chunk of the DNA in the promoter region, typically on the order of 2,000 to 4,000 base pairs, and let the CRISPR scissors alter this part of the genetic code. Then, he and his scientific team chose which cuts from the scissors and subsequent repairs by the cell’s machinery gave the desired modifications to the traits they were studying.

Invented only five years ago, CRISPR is a genetic editing technique that uses tools bacteria have developed to fight off viral infections. Once a bacteria is attacked by a virus, it inserts a small piece of the viral gene into its own sequence. If a similar virus attacks again, the bacteria immediately recognizes the invader and cuts the sequence away.

Scientists sometimes use these molecular scissors to trim specific gene sequences in a process called a deletion. They are also working toward ways to take another genetic code and insert a replacement. “Replacement technology is only now starting to become efficient,” Lippman said. Clinical researchers are especially excited about the potential for this technique in treating genetic conditions, potentially removing and replacing an ineffective sequence.

In Lippman’s case, he used the scissors to cut in several places in the promoter regions of the tomato plant. Rather than targeting specific genes, he directed those scissors to change the genome at several places. When he planted the new seeds, he explored their phenotype, or the physical manifestation of their genetic instructions. These phenotypes varied along a continuum, depending on the changes in their genes.

By going backward and then comparing the genes of the altered plants to the original, he could then hone in on the precise changes in the genetic code that enabled that variation. This technique allows for a finer manipulation than turning on or off specific genes in which an organism, in this case a plant, would either follow specific instructions or would go on a transcriptional break, halting production until it was turned on again.

At this point, Lippman has worked with each trait individually but hasn’t done quantitative variation for more than one at a time. “The next question,” he said, “is to do this multitargeting.” He will also use the tool to study how genes are instructed to turn on and off during growth, including exploring the levels and location of expression.

Lippman is talking with agricultural and scientific collaborators and hopes to go beyond the tomato to exploring the application of this approach to other crops. He is working with Dave Jackson, who is also a professor at Cold Spring Harbor Laboratory, on applying this model to corn.

The scientific duo has known each other for 20 years. Jackson taught his collaborator when Lippman was a graduate student at Cold Spring Harbor Laboratory and Jackson was chair of his thesis committee.

They have worked together on and off since Lippman became a faculty member about nine years ago. Last year, the two received a National Science Foundation genome grant to work on using CRISPR to study the effect of changes in promoter regions in their respective plant specialties.

“Unfortunately for us, tomato has a faster life cycle than corn, but we hope to have some results in corn this fall,” Jackson explained in an email. Lippman hopes to continue on the path toward understanding how regulatory DNA is controlling complex traits. “We can use this tool to dissect critical regulatory regions,” he said. “When we create this variation, we can look at how that translates to a phenotypic variation.”

Lippman said he is especially excited about the fundamental biological questions related to plant growth and development. When other scientists or agricultural companies attempt to use this approach, they may run into some challenges, he said. Some plants are “not transformable [genetically] easily.” These plants can be recalcitrant to plant transformation, a step sometimes needed for CRISPR gene editing. Still, it is “likely that CRISPR will work in all organisms,” he said.

Lippman hopes others discuss this technique and see the potential for a system that could help to customize plants. “My hope and my anticipation is that people all over the world will look at this paper and say, ‘Let’s start to try this out in our own systems.’ Hopefully, there will be a grass roots effort to import this tool.”

Organizers of the 3rd annual Genome Engineering: The CRISPR-Cas Revolution event, from left, Maria Jasin, Jonathan Weissman, Jennifer Doudna and Stanley Qi. Photo courtesy of CSHL

By Daniel Dunaief

One day, the tool 375 people from 29 countries came to discuss in late July at Cold Spring Harbor Laboratory may help eradicate malaria, develop treatments for cancer and help understand the role various proteins play in turning on and off genes.

Eager to interact with colleagues about the technical advances and challenges, medical applications and model organisms, the participants in Cold Spring Harbor Laboratory’s third meeting on the CRISPR-Cas9 gene editing system filled the seats at Grace Auditorium.

Jason Sheltzer. Photo from CSHL

“It’s amazing all the ways that people are pushing the envelope with CRISPR-Cas9 technology,” said Jason Sheltzer, an independent fellow from Cold Spring Harbor Laboratory who presented his research on a breast cancer treatment.

The technology comes from a close study of the battle between bacteria and viruses. Constantly under assault from viruses bent on commandeering their genetic machinery, bacteria figured out a way of developing a memory of viruses, sending out enzymes that recognize and destroy familiar invaders.

By tapping into this evolutionary machinery, scientists have found that this system not only recognizes genes but can also be used to slice out and replace an errant code.

“This is a rapidly evolving field and we continue to see new research such as how Cas1 and Cas2 recognize their target, which opens the door for modification of the proteins themselves, and the recent discovery of anti-CRISPR proteins that decrease off-target effects by as much as a factor of four,” explained Jennifer Doudna, professor of chemistry and molecular and cell biology at the University of California at Berkeley and a meeting organizer for the last three years, in an email.

Austin Burt, a professor of evolutionary genetics at the Imperial College in London, has been working on ways to alter the genes of malaria-carrying mosquitoes, which cause over 430,000 deaths each year, primarily in Africa.

“To wipe out malaria would be a huge deal,” Bruce Conklin, a professor and senior investigator at the Gladstone Institute of Cardiovascular Disease at the University of California in San Francisco and a presenter at the conference, said in an interview. “It’s killed millions of people.”

Carolyn Brokowski. Photo by Eugene Brokowski

This approach is a part of an international effort called Target Malaria, which received support from the Bill and Melinda Gates Foundation.

To be sure, this effort needs considerable testing before scientists bring it to the field. “It is a promising approach but we must be mindful of the unintended consequences of altering species and impacting ecosystems,” Doudna cautioned.

In an email, Burt suggested that deploying CRISPR in mosquitoes across a country was “at least 10 years” away.

CSHL’s Sheltzer, meanwhile, used CRISPR to show that a drug treatment for breast cancer isn’t working as scientists had thought. Researchers believed a drug that inhibited the function of a protein called maternal embryonic leucine zipper kinase, or MELK, was halting the spread of cancer. When Sheltzer knocked out the gene for MELK, however, he discovered that breast cancer continued to grow or divide. While this doesn’t invalidate a drug that may be effective in halting cancer, it suggests that the mechanism researchers believed was involved was inaccurate.

Researchers recognize an array of unanswered questions. “It’s premature to tell just how predictable genome modification might be at certain levels in development and in certain kinds of diseases,” said Carolyn Brokowski, a bioethicist who will begin a position as research associate in the Emergency Medicine Department at the Yale School of Medicine next week. “In many cases, there is considerable uncertainty about the causal relationship between gene expression and modification.”

Brokowski suggested that policy makers need to appreciate the “serious reasons to consider limitations on nontherapeutic uses for CRISPR.”

Like so many other technologies, CRISPR presents opportunities to benefit mankind and to cause destruction. “We can’t be blind to the conditions in which we live,” said Brokowski.

Indeed, Doudna recently was one of seven recipients of a $65 million Defense Advanced Research Projects Agency award to improve the safety and accuracy of gene editing.

The funding, which is for $65 million over four years, supports a greater understanding of how gene editing technologies work and monitors health and security concerns for their intentional or accidental misuse. Doudna, who is credited with co-creating the CRISPR-Cas9 system with Emmanuelle Charpentier a scientific member and director of the Max Planck Institute for Infection Biology in Berlin, will explore safe gene editing tools to use in animal models and will specifically target Zika and Ebola viruses.

“Like most misunderstood disruptive technologies, CRISPR outpaced the necessary policy and regulatory discussions,” Doudna explained. The scientific community, however, “continued to advance the technology in a transparent manner, helping to build public awareness, trust and dialogue. As a result, CRISPR is becoming a mainstream topic and the public understanding that it can be a beneficial tool to help solve some of our most important challenges continues to grow.”

Visitors enjoyed a wine and cheese party on the Airslie lawn during the event. Photo from CSHL

Cold Spring Harbor Laboratory plans to host its fourth CRISPR meeting next August, when many of the same scientists hope to return. “It’s great that you can see how the field and scientific community as a whole is evolving,” Sheltzer said.

Doudna appreciates the history of Cold Spring Harbor Laboratory, including her own experiences. As a graduate student in 1987, Doudna came across an unassuming woman walking the campus in a tee-shirt: Nobel Prize winner Barbara McClintock. “I thought, ‘Oh my gosh, this is someone I revere,” Doudna recalled. “That’s what life is like” at the lab.

Brokowski also plans to attend the conference next year. “I’m very interested in learning about all the promises CRISPR will offer,” she said. She is curious to see “whether there might be more discussion about ethical and regulatory aspects of this technology.”