You’ve probably heard the term CRISPR in recent years. It is a relatively new breakthrough in science that has applications in human health and even in the fight against HLB. Nian Wang, a professor of microbiology and cell science with the University of Florida Institute of Food and Agricultural Sciences (UF/IFAS), has been on the forefront of CRISPR research. Here he breaks down the technology and its applications to citrus breeding.
Q: What is CRISPR?
A: Scientifically, CRISPR stands for clustered regularly interspaced short palindromic repeats. It is basically the immune system of bacteria and archaea that cut invading bacteria and plasmid DNA. It is the foundation for the CRISPR-Cas genome-editing technology, which we often simply refer it as the CRISPR technology, CRISPR genome editing or CRISPR. It acts like molecular scissors that can be used to cut a specific piece of DNA. In the context of developing disease-resistant (especially HLB disease) citrus varieties, it is often used to remove part of the disease susceptibility genes to deactivate it or make it not respond to the pathogens. Consequently, plants will become disease resistant or tolerant.
Q: How have you been using CRISPR in your research in the fight against HLB?
A: We have been using CRISPR to edit citrus genes to improve citrus resistance/tolerance against HLB. Because the HLB pathogen Candidatus Liberibacter asiaticus (CLas) has not been cultured in media, it has been difficult to pinpoint the right target genes for editing. Our recent study shows that HLB is a pathogen-triggered immune disease. Immune responses are generally good for plants or humans against pathogen infections. However, when the immune response is always turned on, it will have significant negative consequences as it does in many autoimmune diseases.
In short, we have been using CRISPR to manipulate citrus immune response to generate HLB-tolerant/resistant citrus in several ways:
- Editing HLB susceptibility genes to reduce the growth of CLas in citrus or reduce HLB damage
- Editing genes related to antioxidants to mitigate oxidative stress caused by CLas
- Editing genes in the immune-signaling pathway specifically triggered by CLas, such as genes responsible for reactive oxygen species production.
Q: How long have you been working on this CRISPR research?
A: It has been a long journey. We have been working on CRISPR since early 2013. We actually started working on genome editing in 2012 using a different technology called TALEN. But the TALEN technology is more time-consuming, expensive and complicated to work with, so we quickly switched to CRISPR once it showed promise in editing human cells in January 2013.
We have been developing and optimizing the CRISPR genome-editing technology for citrus since then. We were finally able to generate transgene-free genome-edited citrus plants in 2022. That means genetically modified organism (GMO)-free. It is important to note that transgenic crops face many challenges to be used in production because of regulations and public perception concerns, even though there are multiple GMO crops such as corn and soybean.
We have been dreaming about developing transgene-free genome-editing technologies for citrus from day one. It took us about 10 years to get there. We were excited once we finally developed the transgene-free CRISPR genome-editing technology for citrus in 2022.
Importantly, regulatory agencies have finalized rules that make it easy to deregulate transgene genome-edited crops generated by the CRISPR technology. As for application of the CRISPR technology in the fight against HLB, this really gives us a powerful tool to speed up the process to generate HLB-resistant/tolerant citrus varieties.
Q: From start to finish, roughly how long does it take to develop a CRISPR citrus tree?
A: The editing starts at the single cell level, and the regeneration into plantlets takes about 10 months. It needs another 12 to 18 months for propagation to get enough replicates, to make plants grow bigger for CLas inoculation. We also need to make sure that the edited plants are not only resistant or tolerant to HLB, but also have the least negative effects on other traits such as fruit yield and quality.
So, it is roughly one year to generate the CRISPR tree. But it takes four to six years to complete multiple evaluations and tests and go through the regulation process at federal and state levels before reaching the hands of citrus growers as registered cultivars.
Q: How many targets have you identified that you believe have potential in resisting HLB?
A: We have been working on approximately 40 targets. Because CLas has not been cultured in artificial media, we have not been able to narrow down the target genes to single genes like with canker. We have seven or eight genes that we feel are most promising. We have generated over 100 sweet orange lines for those targets. We have mostly focused on sweet orange, especially Hamlin, because sweet orange is the main cultivar in Florida.
We need to focus on one cultivar first. We can edit other cultivars once we know the target for sure. We think this is the most efficient way to go forward. Most of our current edited lines are transgenic with few being transgene-free. Most of our transgene-free edited lines remain in the petri-dish stage. But our transgenic CRISPR lines will be tested in the field in March 2024. I think multiple lines will be HLB resistant or tolerant. Of course, we need to collect data to verify that, and we need to make sure there are no significant negative effects from the editing.
Q: Is there anything else you would like to add?
A: I would like to thank my many UF/IFAS colleagues who have collaborated in this research. The progress we’ve made couldn’t happen without their help. I want to thank the Graves family for their support of the Graves Eminent Scholar Endowed Chair, which I am honored to hold. This endowment is used for citrus biotechnology to enhance citrus research and the citrus industry. This endowment will provide the much-needed funds to speed up and scale up the generation of HLB-resistant/tolerant citrus varieties using the CRISPR technology. In addition, it allows the exploration of high-risk, high-reward research activities.
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