Transgene-free genome editing in citrus and poplar meristem tissues via biolistic ribonucleoprotein delivery of CRISPR-Cas9.
Rocha DC, Omoregbee MO, Luo W, Fang H, Ye Q
Summary
7.8/10Researchers developed a method to edit genes directly in woody plants like citrus and poplar without inserting foreign DNA, using a new delivery technique that targets regenerative tissues in the plants' growing regions. This breakthrough solves a major bottleneck in plant biotechnology, since many tree species are difficult to genetically modify using traditional methods.
Key Findings
Biolistic delivery of CRISPR-Cas9 ribonucleoproteins successfully generated targeted mutations in meristem tissues (CsNPR3 locus in citrus, Pt4CL1 gene in poplar)
RNP-based approach achieved genome editing in citrus where plasmid-based vectors completely failed
Transgene-free edited plants were produced, eliminating regulatory complications from foreign DNA sequences
Original Abstract
Biolistic particle bombardment was used to deliver CRISPR-Cas9 ribonucleoprotein complexes (RNP) into the shoot apical meristem tissue of citrus and axillary meristem tissue of poplar, generating directed mutations in target genes. The use of meristematic tissues offers a strategic approach to genome editing in woody species, especially those that are recalcitrant to conventional tissue culture, as these regions contain totipotent, highly regenerative cells capable of giving rise to whole plants. Here, we employed biolistic delivery of genome-editing reagents into theshoot apical meristem (SAM) of citrus and the axillary meristems (AXM) of poplar. The system was first validated using a GFP expression construct and subsequently applied for targeted genome editing. In citrus, edited plants were obtained at the CsNPR3 locus exclusively through the delivery of CRISPR/Cas9 ribonucleoproteins (RNPs), whereas plasmid-based vectors were unsuccessful. Similarly, genome editing in poplar was achieved using RNPs targeting the Pt4CL1 gene. Although chimeric events were detected, this approach provides a feasible and innovative framework for producing transgene-free edited perennial plants.
Citrus is a genus of flowering trees and shrubs in the family Rutaceae. Plants in the genus produce citrus fruits, such as citrons, mandarins, and pomelos. Many important citrus crops have been developed through extensive hybridization, including oranges, lemons, grapefruits, and limes, all of wh...
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