Transgene-Free Canker-Resistant Citrus sinensis with Cas12/RNP

Jahnavee Palsodkar, Institute of Bioinformatics and Biotechnology, Savitribai Phule Pune University, Pune, Maharashtra, India

Battling pathogens for healthy fruits

Citrus canker is a pathogenic disease that impacts citrus plants, inducing lesions on various plant organs such as leaves and fruits. This severely diminishes the viability and economic value of the plant. Efforts to cultivate a resistant citrus variety for commercial purposes have been largely unsuccessful, primarily due to the prolonged breeding period and the stringent regulations pertaining to transgenic plants. However, a promising breakthrough has emerged. In their 2023 study, Su et al. introduced a noteworthy development: a transgene-free, canker-resistant strain of Citrus sinensis (commonly known as Orange). Remarkably, they achieved this accomplishment in just 10 months, all while preserving the economically significant phenotype of the wild-type plant.

The pathogen vs. The remedy

Citrus canker arises due to Xanthomonas citri sbsp. citri (hereafter referred to as Xcc). An Xcc-encoded pathogenic element activates the canker-susceptibility gene LOB1 in citrus plants, inducing to the manifestation of citrus canker symptoms. In this study, the researchers used the Cas12aU RNA ribonucleoprotein (RNP) complex to disrupt an exon within the coding sequence of the CsLOB1 gene in the embryogenic protoplasts of C. sinensis. This imparts Xcc resistance to the subsequently regenerated plant.

The strategy

Initially, to assess the efficiency of the RNP system i.e. the LbCas12aU/crRNA complex, the authors performed targeted editing of the CsPDS gene. This gene was chosen due to its easily detectable albino phenotype. The editing efficiency was gauged through in-vitro digestion of the first exon of the CsPDS gene. With successful results, the study progressed to the transformation of embryogenic C. sinensis protoplasts via the PEG method. Embryos exhibiting the albino phenotype were subjected to Sanger sequencing, which unveiled a consistent 9 base pair mutation at the designated site. Based on these positive results, the authors advanced to the targeted mutation of the CsLOB1 gene.

Mechanism

The researchers utilized the crRNA, a component of the guide RNA that is complementary to the target DNA, to specifically target the second exon in the coding region of the CsLOB1 gene. They designed it meticulously to prevent mutations in homologous sites outside of the intended target. They achieved the transformation of embryogenic protoplasts through the utilization of the LbCas12aU/crRNA complex. Out of the 38 lines that survived grafting, 97.4% exhibited homozygous/biallelic mutations. Subsequent NGS analysis uncovered that all lines carried an identical 7-base pair mutation at the specified site and contained no off-target mutations.

Conclusion

Further evaluation of the CsLOB1 mutants showed phenotype of narrower leaves in comparison to the wild-type in 6 out of the 38 lines. This phenomenon can be attributed to somaclonal variations arising due to tissue culture. Infecting these mutants with Xcc did not trigger any canker symptoms. Additionally, the mutants displayed considerably lower Xcc titres compared to the similarly infected wild-type C. sinensis. To study the mechanism behind the Xcc resistance in these mutants, qRT-PCR analysis was conducted on genes recognized to be upregulated by LOB1 during Xcc infections. The findings showcased a notably reduced expression of these genes as compared with the wild-type, indicating that the mutation in the LOB1 gene had effectively halted the activation of downstream genes responsible for causing citrus canker.

Summary

To recapitulate, Su et al. successfully developed an Xcc-resistant version of Citrus sinensis utilizing the transgene-free RNP technique. This variety is devoid of any off-target mutations or foreign genetic elements, and it presents no discernible deviations from the wild-type strains apart from its heightened resistance to canker. These attributes played a pivotal role in securing it a regulatory endorsement from the USDA APHIS (Animal and Plant Health Inspection Service), marking a significant milestone in overcoming a crucial barrier towards its viable commercial deployment.

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Reference:

Su, H., Wang, Y., Xu, J. et al. Generation of the transgene-free canker-resistant Citrus sinensis using Cas12a/crRNA ribonucleoprotein in the T0 generation. Nat Commun 14, 3957 (2023). https://doi.org/10.1038/s41467-023-39714-9

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