What Is CRISPR and What Does it Mean for Citrus?

By Fred Gmitter, Yi Zhang and Jude Grosser

It is very likely that you have heard about the use of CRISPR technology and its great potential for addressing human health issues, as well as the promise it holds for providing solutions for major agricultural challenges, particularly for huanglongbing (HLB) in citrus. Granting agencies are supporting many citrus research projects aimed at developing resistance to HLB, through genome editing using CRISPR technology.

Although there is public awareness of the technology, and CRISPR has become a part of the citrus industry’s vocabulary, there are quite a few questions about this subject that we hope to answer for you in a simple and understandable way in this article. What is CRISPR and how does it work? What kind of objectives can be addressed, what is necessary for successful application of the technology and what are the limitations? How is it different from GMO (genetically modified organism) technology? Finally, what are the prospects for global commercialization of citrus products developed using genome editing technology?

CRISPR EXPLAINED
The most widely used genome editing system is CRISPR-Cas9, which stands for clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9. This is a short piece of bacterial DNA that is part of the defense mechanism used to fight invading viruses, by recognizing them and “cutting” their DNA or RNA.

CRISPR

Figure 1. This simplified diagram shows how CRISP/Cas9 works to edit the genome. The guide RNA is based on the DNA sequence of the target gene. This guide is complexed with the Cas9 protein, which cuts the host DNA. Then, changes are made to the gene either by deleting or adding a few nucleotides to the gene sequence. This changes the “spelling” of the gene, thereby also altering its function.

By clever molecular biology techniques, this bacterial defensive machinery has been used now to make small changes in the DNA sequences of genomes of many different organisms, including fungi, plants, animals and even humans (Figure 1). The concept has been evolving since the late 1980s when such bacterial sequences were first identified. But it wasn’t until just five years ago, in 2013, when the first use of CRISPR for editing eukaryotic cells, including those from plants, was reported. In 2014, Nian Wang’s lab at the University of Florida Institute of Food and Agricultural Sciences Citrus Research and Education Center first published on successfully editing the citrus genome using Cas9 (Figure 2).

CRISPR

Figure 2. CRISPR technology has been under development since the late 1980s. It was first demonstrated to be effective in citrus in 2014. Since then, many research projects have been initiated to use CRISPR in hopes of making HLB-resistant plants.

To target any specific gene in the citrus genome that we wish to affect, the exact DNA sequence of that gene must be known. Until now, most citrus researchers relied on the publicly available reference genomes, such as the Clementine mandarin that was developed by the International Citrus Genome Consortium, to find the full sequences of their targeted gene.

An RNA “guide” molecule is then produced based on the specific gene sequence and complexed with the Cas9 protein, to find the gene’s exact location in the genome. Once the complex is aligned with the gene, Cas9 does its job by cutting both strands of the DNA molecule near that location. These cuts then can be repaired, and the strands rejoined, but with either some missing or some additional DNA sequence to change the “spelling” of the gene. By doing so, a given gene can be made to be non-functional or to have its function and/or expression changed in some way different from the natural condition. The simple idea for making a susceptible plant resistant to a pathogen is to target the host gene that responds to something from the pathogen, some receptor for example, that leads then to the development of disease in the plant. Knock out the receptor and you prevent the disease.

CHALLENGES AND LIMITATIONS
Sounds simple, but it can be far more difficult in practice, particularly with a complicated disease such as HLB or other aspects of the plant that are not under the control of a single gene, but rather are influenced by many different genes. The greatest challenge to solving HLB through genome editing is the identification of the most likely specific target.

Much research has taken place to understand the mechanism of the disease in citrus plants, by studying gene expression in tolerant and more HLB-sensitive citrus varieties in leaves, stems, fruit and roots. Researchers also have used microscopy to visualize what is happening within the phloem tissues. They have measured enzyme activity in various important metabolic pathways in the plants. They are comparing apparent tolerant mutants with their normal types, looking for clues. But because HLB is associated with many changes in infected citrus trees, it has been very difficult to identify the most likely genes to target.

A further complication to solving the HLB problem by genome editing is that although the Clementine genome sequence is of very high quality, and there are several others also publicly available, none of these genomes are complete and perfect. So, when researchers look for the full and complete sequence of their potential gene target, they may not find all that is needed to design an effective RNA guide molecule.

These sequences were produced using what was the state of the art several years ago, but now with new sequencing and genome assembly tools, nearly perfect and complete genome sequences are closer to reality. It will be of great value to the effort to have these new genome technologies applied to our most common citrus varieties as well, so we can target precisely what we need to edit within their genomes. The sequence of the target gene may be different in oranges than it is in Clementine, grapefruit, etc.

There are other challenges and limitations to the technology, in addition to the central problem of identifying the relevant targets and having their full and correct sequences. CRISPR does rely on genetic transformation to get the complex into the plant and to have it perform its functions. This can be more difficult to do than standard genetic transformations to inset foreign DNA into the plants. In so doing, a consequence with some constructs used for CRISPR is that they still carry some of the telltale signatures of standard GMOs, such as antibiotic-resistant genes or the green fluorescent protein used for selection of modified plants.

So, researchers are working in citrus and with many other plant systems to develop better constructs and new approaches to improve the efficiency of transformation, and to remove all of the extra baggage of the construct. The end goal is that there is nothing else at all in the plant genome except the plant’s own original DNA and the changed “spelling” that codes for the gene of interest.

And because many genes are members of gene families with very similar sequences through much of their structure, it is possible that the guide can be less than specific and target other areas of the genome that are not intended. However, compared with traditional gene knock-out methods, CRISPR is superior in that it can aim at multiple gene targets in one go so it may greatly shorten the time needed to modify multiple genes to achieve desirable results — for instance, the resistance to HLB. Because there is a great deal of research into tool development outside of the citrus research community, the CRISPR system is constantly being upgraded and improved, and many of these technical limitations are being resolved.

GENOME EDITING VS. GMOS
Genome editing differs from standard GMO plants in a very fundamental way. GMOs involve the incorporation of foreign DNA from other organisms such as bacteria, viruses, other plants, even insects and animals. By contrast, genome editing can be accomplished in such a way that there is absolutely nothing foreign that remains in the plant, only its own natural DNA sequences with a small change in the “spelling.”

Such changes in the DNA code are very common and happen all the time, naturally. If you were to have your own genome sequenced multiple times, and you used different cells from your bones, skin, muscles, etc. each time, you would find that there are literally hundreds to thousands of single letter changes to your own genome. This could be tremendously significant as the regulatory agencies consider how to deal with edited plants and as they have their day in the court of public opinion.

Already, CRISPR-edited, non-browning mushrooms have been developed. The U.S. Department of Agriculture made the decision that these mushrooms are not subject to regulation, as would be a GMO mushroom with the same trait. It will be important, not only for the citrus industry and its consumers, but for all of agriculture and its ability to feed the ever-expanding human population on our planet, that a rational approach to this technology evolves.

As citrus scientists come to understand better the genetic control, not only of disease resistance or susceptibility, but of the many other important traits and characteristics (such as citrus fruit and juice color, flavor, quality, abiotic stress tolerance and seediness), genome editing holds great promise for targeted improvements of citrus. That is not to say that it can or will replace traditional breeding approaches entirely, because as stated above, many important characteristics of citrus trees are under the control of a large number of genes and their interactions. Traditional plant breeding techniques have a long and proven record of success for such goals and objectives. However, genome editing by CRISPR and other emerging techniques provide powerful new tools in the plant breeders’ tool box and might speed up the breeding process.

Fred Gmitter and Jude Grosser are professors, and Yi Zhang is a post-doctoral research associate — all at the University of Florida Institute of Food and Agricultural Sciences Citrus Research and Education Center in Lake Alfred.

 

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