Taking a Closer Look at CRISPR in Citrus Breeding

By Manjul Dutt

Editor’s Note: This is the second article in a series outlining how genetic technology can be using in citrus breeding.

Traditional methods for improving citrus, such as crossbreeding different varieties, can be time-consuming due to the creation of a random mix of genes from both parents through a process called genetic introgression. In the article last month, we examined genetic tools such as genetically modified organisms and how they compare to newer technology like CRISPR. This month, we take a closer look at CRISPR. Understanding the complexities of genetic transformation and CRISPR is essential for developing innovative solutions in biotechnology, such as creating genetically modified crops and implementing strategies against HLB.

DNA EDITING USING CRISPR

A more precise method of improving plants is called DNA/gene editing or CRISPR. Instead of adding new genes, gene editing makes tiny changes to the plant’s existing DNA. This was discovered from a natural defense system in bacteria. Bacteria use CRISPR to remember viruses that have attacked them, so they can fight back in the future. Scientists realized they could use this system to make very precise changes to the DNA of plants, animals and even humans. CRISPR works by using a special protein that acts like a pair of scissors. It’s guided to the exact spot in the DNA. Once there, it cuts the DNA so scientists can remove, fix or insert new genetic material.

Gene editing is exciting because:

• It can remove unwanted traits (like genes that make trees vulnerable to disease).
• It doesn’t always involve adding foreign DNA, which makes it less controversial.
• It’s very precise and faster than older methods.
• Some gene-edited citrus trees are already in the testing phase and show promise.

CRISPR TIMELINE

CRISPR-edited trees can be created using either the genetic transformation process (for a proof-of-concept study) or the CRISPR RNP-mediated genome editing process. While trees produced through the genetic transformation process are classified as genetically modified organisms (GMOs), those created using the RNP-mediated method result in non-GM plants. If citrus protoplasts are used to edit DNA using the RNP-mediated method, the resulting plants will be juvenile and take five to seven years to flower and bear fruit.

The overall process of developing a non-GM CRISPR-edited citrus plant is:

1. Target Identification, Guide RNA Design and RNP Complex Formation (1 year)
Scientists identify a specific gene in the citrus tree’s genome that they want to edit, knock out or modify, such as to enhance the tree’s tolerance to huanglongbing (HLB). They then design a guide RNA (gRNA) that directs the Cas protein to the exact location in the DNA. Once the gRNA is synthesized, it is combined with the Cas protein — acting like molecular scissors — to form a ribonucleoprotein (RNP) complex. This complex is pre-assembled in the lab without involving any DNA or vectors, making it a transgene-free editing method.

2. Delivery of RNPs into Citrus Cells (1-2 years)
Due to the complex biology of citrus trees, manipulating them genetically is challenging. Currently, RNPs are delivered into protoplasts — plant cells that lack cell walls — using a method similar to the one used in traditional GM production processes. Once the RNP complex is inside the cell, it locates the target DNA. The Cas protein then makes a precise cut at the target site. The cell attempts to repair this cut, which often results in small insertions or deletions that disrupt or modify the gene.

3. Regeneration of Whole Plants (1-2 years)
Edited cells are regenerated into plantlets through tissue culture, where hormones are used to promote the development of shoots and roots. This is a crucial and time-consuming step in citrus regeneration, as it can take a long time for plants to develop from protoplasts. Molecular analysis is performed to confirm the editing process. Those plants that have been successfully edited are then transferred to the greenhouse for further growth and development.

3. Testing and Regulatory Considerations (3-8 years)
If successful, the edited trees are either grown in a greenhouse for HLB evaluation or subjected to field trials to assess their performance. Since no foreign genes are utilized, RNP-edited trees may be exempt from GM regulations in certain countries, such as the United States. However, this exemption varies worldwide. The certification and commercialization progress generally follows that explained before.

ENSURING GM CITRUS SAFETY

Before any GM citrus trees are allowed in commercial groves, they go through strict testing. Scientists conduct extensive lab, greenhouse and field testing to make sure:

• The new trees grow normally and produce juice similar to non-modified controls.
• The fruit is safe to eat.
• The trees don’t harm the environment.

Regulatory agencies such as the U.S. Department of Agriculture, Food and Drug Administration and Environmental Protection Agency in the United States, along with similar organizations worldwide, review these studies. GM citrus is deemed safe once it has passed all regulatory assessments.

Manjul Dutt is an assistant professor at the University of Florida Institute of Food and Agricultural Sciences Citrus Research and Education Center in Lake Alfred.