Engineering CRISPR immune systems conferring GLRaV-3 resistance in grapevine
Bolei Jiao1, Xinyi Hao1, Zhiming Liu1
1State Key Laboratory of Crop Stress Biology in Arid Areas, College of Horticulture, Northwest A&F University, Yangling, Shaanxi 712100, China.
Horticulture Research
|January 18, 2022
Summary
CRISPR technology using FnCas9 and LshCas13a effectively inhibits Grapevine leafroll-associated virus 3 (GLRaV-3). This study demonstrates potential for engineering virus-resistant grapevines against GLRaV-3 and other RNA viruses.
Area of Science:
- Plant Pathology
- Molecular Biology
- Biotechnology
Background:
- Grapevine leafroll disease (GLD), caused by Grapevine leafroll-associated virus 3 (GLRaV-3), significantly reduces grapevine yields globally.
- Developing virus-resistant grapevines is crucial for disease management, but natural resistance is lacking in the Vitis genus.
- Research on anti-GLRaV-3 strategies in grapevines has been limited.
Purpose of the Study:
- To establish an effective screening system for transgenic constructs targeting GLRaV-3 in grapevine.
- To evaluate the efficacy of CRISPR/FnCas9 and LshCas13a in inhibiting GLRaV-3.
- To identify optimal constructs for developing GLRaV-3-resistant grapevines.
Main Methods:
- Development of a highly effective transgenic construct screening system using an optimized Agrobacterium-mediated transient delivery system in grapevine plantlets.
- Expression of FnCas9 and LshCas13a to target and inhibit GLRaV-3.
- Evaluation of viral interference efficiency based on Cas gene expression and RNA binding activity.
Main Results:
- CRISPR/FnCas9 and LshCas13a demonstrated significant GLRaV-3 inhibition.
- Three vectors (pCR01-CP, pCR11-Hsp70h, and pCR11-CP) showed the most robust inhibition efficiency.
- FnCas9's efficiency correlated with its RNA binding activity and Cas gene expression levels.
- LshCas13a exhibited superior interference efficiency against viruses compared to FnCas9.
Conclusions:
- RNA-targeting CRISPR mechanisms, specifically FnCas9 and LshCas13a, can confer immunity against GLRaV-3 in grapevines.
- These systems offer promising new strategies for controlling GLRaV-3 and other RNA viruses in fruit crops.
- The identified vectors provide a foundation for engineering GLRaV-3-resistant grapevine varieties.
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