Generation of transgene-free canker-resistant Citrus sinensis cv. Hamlin in the T0 generation through Cas12a/CBE co-editing

Jia, Hongge; Omar, Ahmad A.; Xu, Jin; Dalmendray, Javier; Wang, Yuanchun; Feng, Yu; Wang, Wenting; Hu, Zhuyuan; Grosser, Jude W.; Wang, Nian

doi:10.3389/fpls.2024.1385768

Cited by 4 publications

References 77 publications

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Development and application of gene editing in citrus: how and what to do

Wang,

Zhao,

et al. 2024

HORTIC. ADV.

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Conventional breeding techniques have been effectively utilized for the enhancement of citrus varieties. Nonetheless, traits such as an extended juvenile phase, cross- or self-incompatibility, high genetic heterozygosity, and polyembryony have posed significant challenges and limitations to these methods. The clustered regularly interspaced short palindromic repeats (CRISPR) genome editing has progressively emerged as a vital tool for citrus breeding and research. This article reviews the array of CRISPR/Cas genome editing systems, emphasizes recent advancements in citrus genome editing using CRISPR/Cas, and explores the application of this technology to bolster resistance to citrus canker. The review also covers the development of CRISPR/Cas-mediated transformation and regeneration systems for citrus, alongside approaches for generating transgene-free citrus germplasm. Moreover, the regulatory landscape and societal acceptance of CRISPR/Cas genome editing are examined. Lastly, potential applications of genome editing in citrus breeding are proposed, with attention to prospective challenges.

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Development and application of gene editing in citrus: how and what to do

Wang,

Zhao,

et al. 2024

HORTIC. ADV.

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Cas12a/3 crRNAs RNP transformation enables transgene-free multiplex genome editing, long deletions, and inversions in citrus chromosome in the T0 generation

Su,

Wang,

et al. 2024

Preprint

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Citrus canker, caused byXanthomonas citrisubsp. citri (Xcc), is a devastating disease worldwide. Previously, we successfully generated canker-resistantCitrus sinensiscv. Hamlin lines in the T0 generation, achieving a mutation efficiency of 97.4%. This was achieved through the transformation of embryogenic protoplasts using the Cas12a/1 crRNA ribonucleoprotein (RNP) system to edit the canker susceptibility gene,CsLOB1, which led to small indels. Here, we transformed embryogenic protoplasts of Hamlin with Cas12a/3 crRNAs RNP, resulting in 100% efficiency in editing theCsLOB1gene in the T0 generation. Among the 10 transgene-free genome-edited lines, long deletions were obtained in five lines. Additionally, inversions were observed in three of the five edited lines with long deletions, but not in any edited lines with short indel mutations, suggesting long deletions are required for inversions. Biallelic mutations were observed for each of the three target sites in 4 of the 10 edited lines when 3 crRNAs were used, demonstrating that transformation of embryogenic citrus protoplasts with Cas12a/3 crRNAs RNP can be very efficient for multiplex editing. Our analysis revealed the absence of off-target mutations in the edited lines. Thesecslob1mutant lines were canker-resistant and no canker symptoms were observed after inoculation withXccandXccgrowth was significantly reduced in thecslob1mutant lines compared to the wild type plants. Taken together, Cas12a/3 crRNAs RNP transformation of embryogenic protoplasts of citrus provides a promising solution for transgene-free multiplex genome editing with high efficiency and for deletion of long fragments.

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Cas12a RNP-mediated co-transformation enables transgene-free multiplex genome editing, long deletions, and inversions in citrus chromosome

Su,

Wang,

et al. 2024

Front. Plant Sci.

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IntroductionCitrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a devastating disease worldwide. Previously, we successfully generated canker-resistant Citrus sinensis cv. Hamlin lines in the T0 generation. This was achieved through the transformation of embryogenic protoplasts using the ribonucleoprotein (RNP) containing Cas12a and one crRNA to edit the canker susceptibility gene, CsLOB1, which led to small indels.MethodsHere, we transformed embryogenic protoplasts of Hamlin with RNP containing Cas12a and three crRNAs.ResultsAmong the 10 transgene-free genome-edited lines, long deletions were obtained in five lines. Additionally, inversions were observed in three of the five edited lines with long deletions, but not in any edited lines with short indel mutations, suggesting long deletions maybe required for inversions. Biallelic mutations were observed for each of the three target sites in four of the 10 edited lines when three crRNAs were used, demonstrating that transformation of embryogenic citrus protoplasts with Cas12a and three crRNAs RNP can be very efficient for multiplex editing. Our analysis revealed the absence of off-target mutations in the edited lines. These cslob1 mutant lines were canker- resistant and no canker symptoms were observed after inoculation with Xcc and Xcc growth was significantly reduced in the cslob1 mutant lines compared to the wild type plants.DiscussionTaken together, RNP (Cas12a and three crRNAs) transformation of embryogenic protoplasts of citrus provides a promising solution for transgene-free multiplex genome editing with high efficiency and for deletion of long fragments.

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Generation of transgene-free canker-resistant Citrus sinensis cv. Hamlin in the T0 generation through Cas12a/CBE co-editing

Cited by 4 publications

References 77 publications

Development and application of gene editing in citrus: how and what to do

Development and application of gene editing in citrus: how and what to do

Cas12a/3 crRNAs RNP transformation enables transgene-free multiplex genome editing, long deletions, and inversions in citrus chromosome in the T0 generation

Cas12a RNP-mediated co-transformation enables transgene-free multiplex genome editing, long deletions, and inversions in citrus chromosome

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