Tag Archives: Huanglongbing (HLB)

The CRISPR yogurt story and a hornless cattle update

Clustered regularly interspaced short palindromic repeats (CRISPR) does not and never has made much sense to me. I understand each word individually it’s just that I’ve never thought they made much sense strung together that way. It’s taken years but I’ve finally found out what the words (when strung together that way) mean and the origins for the phrase. Hint: it’s all about the phages.

Apparently, it all started with yogurt as Cynthia Graber and Nicola Twilley of Gastropod discuss on their podcast, “4 CRISPR experts on how gene editing is changing the future of food.” During the course of the podcast they explain the ‘phraseology’ issue, mention hornless cattle (I have an update to the information in the podcast later in this posting), and so much more.

CRISPR started with yogurt

You’ll find the podcast (almost 50 minutes long) here on an Oct. 11, 2019 posting on the Genetic Literacy Project. If you need a little more encouragement, here’s how the podcast is described,

To understand how CRISPR will transform our food, we begin our episode at Dupont’s yoghurt culture facility in Madison, Wisconsin. Senior scientist Dennis Romero tells us the story of CRISPR’s accidental discovery—and its undercover but ubiquitous presence in the dairy aisles today.

Jennifer Kuzma and Yiping Qi help us understand the technology’s potential, both good and bad, as well as how it might be regulated and labeled. And Joyce Van Eck, a plant geneticist at the Boyce Thompson Institute in Ithaca, New York, tells us the story of how she is using CRISPR, combined with her understanding of tomato genetics, to fast-track the domestication of one of the Americas’ most delicious orphan crops [groundcherries].

I featured Van Eck’s work with groundcherries last year in a November 28, 2018 posting and I don’t think she’s published any new work about the fruit since. As for Kuzma’s point that there should be more transparency where genetically modified food is concerned, Canadian consumers were surprised (shocked) in 2017 to find out that genetically modified Atlantic salmon had been introduced into the food market without any notification (my September 13, 2017 posting; scroll down to the Fish subheading; Note: The WordPress ‘updated version from Hell’ has affected some of the formatting on the page).

The earliest article on CRISPR and yogurt that I’ve found is a January 1, 2015 article by Kerry Grens for The Scientist,

Two years ago, a genome-editing tool referred to as CRISPR (clustered regularly interspaced short palindromic repeats) burst onto the scene and swept through laboratories faster than you can say “adaptive immunity.” Bacteria and archaea evolved CRISPR eons before clever researchers harnessed the system to make very precise changes to pretty much any sequence in just about any genome.

But life scientists weren’t the first to get hip to CRISPR’s potential. For nearly a decade, cheese and yogurt makers have been relying on CRISPR to produce starter cultures that are better able to fend off bacteriophage attacks. “It’s a very efficient way to get rid of viruses for bacteria,” says Martin Kullen, the global R&D technology leader of Health and Protection at DuPont Nutrition & Health. “CRISPR’s been an important part of our solution to avoid food waste.”

Phage infection of starter cultures is a widespread and significant problem in the dairy-product business, one that’s been around as long as people have been making cheese. Patrick Derkx, senior director of innovation at Denmark-based Chr. Hansen, one of the world’s largest culture suppliers, estimates that the quality of about two percent of cheese production worldwide suffers from phage attacks. Infection can also slow the acidification of milk starter cultures, thereby reducing creameries’ capacity by up to about 10 percent, Derkx estimates.
In the early 2000s, Philippe Horvath and Rodolphe Barrangou of Danisco (later acquired by DuPont) and their colleagues were first introduced to CRISPR while sequencing Streptococcus thermophilus, a workhorse of yogurt and cheese production. Initially, says Barrangou, they had no idea of the purpose of the CRISPR sequences. But as his group sequenced different strains of the bacteria, they began to realize that CRISPR might be related to phage infection and subsequent immune defense. “That was an eye-opening moment when we first thought of the link between CRISPR sequencing content and phage resistance,” says Barrangou, who joined the faculty of North Carolina State University in 2013.

One last bit before getting to the hornless cattle, scientist Yi Li has a November 15, 2018 posting on the GLP website about his work with gene editing and food crops,

I’m a plant geneticist and one of my top priorities is developing tools to engineer woody plants such as citrus trees that can resist the greening disease, Huanglongbing (HLB), which has devastated these trees around the world. First detected in Florida in 2005, the disease has decimated the state’s US$9 billion citrus crop, leading to a 75 percent decline in its orange production in 2017. Because citrus trees take five to 10 years before they produce fruits, our new technique – which has been nominated by many editors-in-chief as one of the groundbreaking approaches of 2017 that has the potential to change the world – may accelerate the development of non-GMO citrus trees that are HLB-resistant.

Genetically modified vs. gene edited

You may wonder why the plants we create with our new DNA editing technique are not considered GMO? It’s a good question.

Genetically modified refers to plants and animals that have been altered in a way that wouldn’t have arisen naturally through evolution. A very obvious example of this involves transferring a gene from one species to another to endow the organism with a new trait – like pest resistance or drought tolerance.

But in our work, we are not cutting and pasting genes from animals or bacteria into plants. We are using genome editing technologies to introduce new plant traits by directly rewriting the plants’ genetic code.

This is faster and more precise than conventional breeding, is less controversial than GMO techniques, and can shave years or even decades off the time it takes to develop new crop varieties for farmers.

There is also another incentive to opt for using gene editing to create designer crops. On March 28, 2018, U.S. Secretary of Agriculture Sonny Perdue announced that the USDA wouldn’t regulate new plant varieties developed with new technologies like genome editing that would yield plants indistinguishable from those developed through traditional breeding methods. By contrast, a plant that includes a gene or genes from another organism, such as bacteria, is considered a GMO. This is another reason why many researchers and companies prefer using CRISPR in agriculture whenever it is possible.

As the Gatropod’casters note, there’s more than one side to the gene editing story and not everyone is comfortable with the notion of cavalierly changing genetic codes when so much is still unknown.

Hornless cattle update

First mentioned here in a November 28, 2018 posting, hornless cattle have been in the news again. From an October 7, 2019 news item on ScienceDaily,

For the past two years, researchers at the University of California, Davis, have been studying six offspring of a dairy bull, genome-edited to prevent it from growing horns. This technology has been proposed as an alternative to dehorning, a common management practice performed to protect other cattle and human handlers from injuries.

UC Davis scientists have just published their findings in the journal Nature Biotechnology. They report that none of the bull’s offspring developed horns, as expected, and blood work and physical exams of the calves found they were all healthy. The researchers also sequenced the genomes of the calves and their parents and analyzed these genomic sequences, looking for any unexpected changes.

An October 7, 2019 UC Davis news release (also on EurekAlert), which originated the news item, provides more detail about the research (I have checked the UC Davis website here and the October 2019 update appears to be the latest available publicly as of February 5, 2020),

All data were shared with the U.S. Food and Drug Administration. Analysis by FDA scientists revealed a fragment of bacterial DNA, used to deliver the hornless trait to the bull, had integrated alongside one of the two hornless genetic variants, or alleles, that were generated by genome-editing in the bull. UC Davis researchers further validated this finding.

“Our study found that two calves inherited the naturally-occurring hornless allele and four calves additionally inherited a fragment of bacterial DNA, known as a plasmid,” said corresponding author Alison Van Eenennaam, with the UC Davis Department of Animal Science.

Plasmid integration can be addressed by screening and selection, in this case, selecting the two offspring of the genome-edited hornless bull that inherited only the naturally occurring allele.

“This type of screening is routinely done in plant breeding where genome editing frequently involves a step that includes a plasmid integration,” said Van Eenennaam.

Van Eenennaam said the plasmid does not harm the animals, but the integration technically made the genome-edited bull a GMO, because it contained foreign DNA from another species, in this case a bacterial plasmid.

“We’ve demonstrated that healthy hornless calves with only the intended edit can be produced, and we provided data to help inform the process for evaluating genome-edited animals,” said Van Eenennaam. “Our data indicates the need to screen for plasmid integration when they’re used in the editing process.”

Since the original work in 2013, initiated by the Minnesota-based company Recombinetics, new methods have been developed that no longer use donor template plasmid or other extraneous DNA sequence to bring about introgression of the hornless allele.

Scientists did not observe any other unintended genomic alterations in the calves, and all animals remained healthy during the study period. Neither the bull, nor the calves, entered the food supply as per FDA guidance for genome-edited livestock.

WHY THE NEED FOR HORNLESS COWS?

Many dairy breeds naturally grow horns. But on dairy farms, the horns are typically removed, or the calves “disbudded” at a young age. Animals that don’t have horns are less likely to harm animals or dairy workers and have fewer aggressive behaviors. The dehorning process is unpleasant and has implications for animal welfare. Van Eenennaam said genome-editing offers a pain-free genetic alternative to removing horns by introducing a naturally occurring genetic variant, or allele, that is present in some breeds of beef cattle such as Angus.

Here’s a link to and a citation for the paper,

Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull by Amy E. Young, Tamer A. Mansour, Bret R. McNabb, Joseph R. Owen, Josephine F. Trott, C. Titus Brown & Alison L. Van Eenennaam. Nature Biotechnology (2019) DOI: https://doi.org/10.1038/s41587-019-0266-0 Published 07 October 2019

This paper is open access.

Citrus canker, Florida, and Zinkicide

Found in Florida orchards in 2005, a citrus canker, citrus greening, poses a serious threat to the US state’s fruit industry. An April 2, 2105 news item on phys.org describes a possible solution to the problem,

Since it was discovered in South Florida in 2005, the plague of citrus greening has spread to nearly every grove in the state, stoking fears among growers that the $10.7 billion-a-year industry may someday disappear.

Now the U.S. Department of Agriculture has awarded the University of Florida a $4.6 million grant aimed at testing a potential new weapon in the fight against citrus greening: Zinkicide, a bactericide invented by a nanoparticle researcher at the University of Central Florida.

An April 2, 2015 University of Central Florida news release by Mark Schlueb (also on EurekAlert), which originated the news item, describes the problem and the solution (Zinkicide),

Citrus greening – also known by its Chinese name, Huanglongbing, or HLB – causes orange, grapefruit and other citrus trees to produce small, bitter fruit that drop prematurely and is unsuitable for sale or juice. Eventually, infected trees die. Florida has lost tens of thousands of acres to the disease.

“It’s a hundred-year-old disease, but to date there is no cure. It’s a killer, a true killer for the citrus industry,” said Swadeshmukul Santra, associate professor in the NanoScience Technology Center at UCF.

The bacteria that causes HLB is carried by the Asian citrus psyllid, a tiny insect that  feeds on leaves and stems of infected citrus trees, then carries the bacteria to healthy trees.

Zinkicide, developed by Santra, is designed to kill the bacteria.

The $4.6 million grant is the largest of five totaling $23 million that were recently announced by the USDA’s National Institute of Food and Agriculture.

The evaluation of Zinkicide is a multi-institute project involving 13 investigators from six institutions. Evan Johnson of UF’s [University of Florida] Citrus Research and Education Center at Lake Alfred is the project director, and there are a dozen co-principal investigators from UF, UCF, Oak Ridge National Laboratory (ORNL), Auburn University, New Mexico State University and The Ohio State University.

”Managing systemic diseases like HLB is a difficult challenge that has faced plant pathologists for many years,” said Johnson “It is a privilege to work with an excellent team of researchers from many different disciplines with the goal of developing new tools that are both effective and safe.”

A portion of the grant money, $1.4 million, flows to UCF, where Santra leads a team that also includes Andre Gesquiere, Laurene Tetard and the Oak Ridge National Laboratory collaborator, Loukas Petridis.

HLB control is difficult because current bactericidal sprays, such as copper, simply leave a protective film on the outside of a plant. The insect-transmitted bacteria bypasses that barrier and lives inside a tree’s fruit, stems and roots, in the vascular tissue known as the phloem. There, it deprives the tree of carbohydrate and nutrients, causing root loss and ultimately death. For a bactericide to be effective against HLB, it must be able to move within the plant, too.

Zinkicide is a nanoparticle smaller than a single microscopic cell, and researchers are cautiously optimistic it will be able to move systemically from cell to cell to kill the bacteria that cause HLB.

“The bacteria hide inside the plant in the phloem region,” Santra said. “If you spray and your compound doesn’t travel to the phloem region, then you cannot treat HLB.”

Zinkicide is derived from ingredients which are found in plants, and is designed to break down and be metabolized after its job is done. [emphasis mine]

It’s the first step in a years-long process to bring a treatment to market. UF will lead five years of greenhouse and field trials on grapefruit and sweet orange to determine the effectiveness of Zinkicide and the best method and timing of application.

The project also includes research to study where the nanoparticles travel within the plant, understand how they interact with plant tissue and how long they remain before breaking down. [emphasis mine]

If effective, the bactericide could have a substantial role in combatting HLB in Florida, and in other citrus-producing states and countries. It would also likely be useful for control of other bacterial pathogens infecting other crops.

The Zinkicide project builds as a spinoff from previous collaborations between Santra and UF’s Jim Graham, at the Citrus Research and Education Center to develop alternatives to copper for citrus canker control.

The previous Citrus Research and Education Foundation (CRDF)-funded Zinkicide project has issued three reports, for June 30, 2014, Sept. 30, 2014, and Dec. 31, 2014. This project’s completion date is May 2015. The reports which are remarkably succinct, consisting of two paragraphs, can be found here.

Oddly, the UCF news release doesn’t mention that Zinkicide (although it can be inferred) is a zinc particulate (I’m guessing they mean zinc nanoparticle) as noted on the CRDF project webpage. Happily, they are researching what happens after the bactericide has done its work on the infection. It’s good to see a life cycle approach to this research.