Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is a destructive citrus disease worldwide. Generating disease-resistant cultivars is the most effective, environmentally friendly and economic approach for disease control. However, citrus traditional breeding is lengthy and laborious. Here, we develop transgene-free canker-resistant Citrus sinensis lines in the T0 generation within 10 months through transformation of embryogenic protoplasts with Cas12a/crRNA ribonucleoprotein to edit the canker susceptibility gene CsLOB1. Among the 39 regenerated lines, 38 are biallelic/homozygous mutants, demonstrating a 97.4% biallelic/homozygous mutation rate. No off-target mutations are detected in the edited lines. Canker resistance of the cslob1-edited lines results from both abolishing canker symptoms and inhibiting Xcc growth. The transgene-free canker-resistant C. sinensis lines have received regulatory approval by USDA APHIS and are exempted from EPA regulation. This study provides a sustainable and efficient citrus canker control solution and presents an efficient transgene-free genome-editing strategy for citrus and other crops.

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is an economically important disease in most citrus production regions worldwide. Xcc secretes a transcriptional activator like effector (TALE) PthA4 to bind to the effector binding elements (EBEs) in the promoter region of canker susceptibility gene LOB1 to activate its expression, which in turn causes canker symptoms. Editing the EBE region with Cas9/gRNA has been used to generate canker resistant citrus plants. However, most of the EBE-edited lines generated contain indels of 1–2 bp, which has higher possibility to be overcome by PthA4 adaptation. The adaptation capacity of TALEs inversely correlates with the number of mismatches with the EBE. LbCas12a/crRNA is known to generate longer deletion than Cas9. In this study, we used a temperature-tolerant and more efficient LbCas12a variant (ttLbCas12a), harboring the single substitution D156R, to modify the EBE region of LOB1. We first constructed GFP-p1380N-ttLbCas12a:LOBP, which was shown to be functional via Xcc-facilitated agroinfiltration in Pummelo (Citrus maxima) leaves. Subsequently, we stably expressed ttLbCas12a:LOBP in Pummelo. Eight transgenic lines were generated, with seven lines showing 100% mutations of the EBE, among which one line is homozygous. The EBE-edited lines had the ttLbCas12a-mediated deletions of up to 10 bp. Importantly, the seven lines were canker resistant and no off-targets were detected. In summary, ttLbCas12a can be used to efficiently generate biallelic/homozygous citrus mutant lines with short deletions, thus providing a useful tool for the functional study and breeding of citrus.

The FLNC gene encodes the sarcomeric protein filamin C which plays a central role in cardiomyocytes. Truncating FLNC mutations (stop or frameshift etc.) also cause dilated cardiomyopathy (DCM) and arrhythmogenic right ventricular cardiomyopathy (ARVC). To further understand the exact role of FLNC in DCM, we have generated a human FLNC knockout cell line from the original embryonic stem cell line H9 by CRISPR/Cas9 gene editing technology in this study. The establishment cell line WAe009-A-70 have a compound heterozygous 4 bp deletion/13 bp deletion in the exon 1 of FLNC which resulted in a frameshift in the translation of FLNC. No filamin C protein was detected in cardiomyocytes differentiated from this cell line. Moreover, WAe009-A-70 also expressed pluripotency markers, maintained the ability to differentiate into the three germ layers in vitro and had a normal karyotype.

Agrobacterium-mediated transient expression (AMTE) is an important tool in plant genetics studies and biotechnology. AMTE remains problematic in citrus and many plant species. Previous research has shown that pretreatment of citrus leaves with Xanthomonas citri subsp. citri ( Xcc), which causes citrus canker, significantly improves the AMTE efficacy. Here, we have shown that Xcc promotes AMTE mainly through triggering cell division and upregulating plant cell wall-degrading enzymes. We demonstrate that Xcc improves AMTE via PthA, a transcription activator-like effector known to trigger cell division in citrus, and mutation of pthA4 abolished the promoting effect of Xcc. Mutation of the effector (PthA4)-binding element in the promoter region and coding region of CsLOB1, which is known to be directly activated by PthA4, significantly reduced Xcc promotion of AMTE. Mutation of PthA4 significantly reduced the expression of cell division-related genes ( CDKA, CDKB1-2, and CDKB2-2) compared with wild-type Xcc and the complemented strain. Cell division inhibitor mimosine but not colchicine also significantly decreased Xcc promotion of AMTE. In addition, PthA4 is known to upregulate plant growth hormones auxin (indole-3-acetic acid), gibberellin, and cytokinin, as well as cell wall-degrading enzymes (e.g., cellulase). Exogenous application of indole-3-acetic acid, cytokinin, and cellulase but not gibberellin significantly improved AMTE in leaves of sweet orange, pummelo, Meiwa kumquat, lucky bamboo, and rose mallow. Our study provides a mechanistic understanding of how Xcc promotes AMTE and develops practical measures to improve AMTE via pretreatment with plant hormones (i.e., auxin and cytokinin) and cellulase. [ ] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .