Xcc-facilitated agroinfiltration of sweet orange leaf.

<p>(a) Scheme of the Xcc-facilitated agroinfiltration method. The sweet orange leaf area, circled by a white oval, was first inoculated with an Xcc re-suspension and then treated 8 hours later with agroinfiltration. Four days later, the oval-circled leaf tissue was analyzed. (b) GUS staining to show that Xcc-facilitated agroinfiltration increased GUS expression in sweet orange leaves. Sweet orange leaves were infiltrated with <i>Agrobacterium tumefaciens</i> harboring pCambia1301, which contains a GUS construct. Eight hours before agroinfiltration, leaves were left non-treated (1), treated with tap water (2), or with Xcc (5×10⁸ CFU/ml) re-suspended in sterile tap water (3). Four days after agroinfiltration, GUS staining was carried out to assay GUS expression. (c) Quantitative GUS assay performed to confirm that Xcc-facilitated agroinfiltration enhanced GUS expression in sweet orange leaves. pCambia1301-transformed <i>Agrobacterium</i> was infiltrated into sweet orange leaves. Eight hours before agroinfiltration, leaves were left non-treated (1), treated with tap water (2), or with Xcc (5×10⁸ CFU/ml) re-suspended in sterile tap water (3). Four days after agroinfiltration, GUS activity was quantified. The experiment was repeated three times. The error bars indicate standard deviations (SD).</p

Targeted genome engineering in sweet orange using the Cas9/sgRNA system.

<p>(a) Scheme of the binary vectors 1380-Cas9 and 1380-Cas9:sgRNA. A Flag tag and a nuclear localization signal (NLS) were fused to the Cas9 N-terminus and C-terminus, respectively. Cas9 catalyzes the cleavage of the sgRNA-targeting sequence immediately upstream of the PAM. Here, Cas9/sgRNA was employed to target the <i>CsPDS</i> gene (red). The <i>Mfe</i>I restriction site and the protospacer adjacent motif (PAM) are underlined. (b) Selective PCR amplification of mutagenized <i>CsPDS</i> genes was used to detect the Cas9/sgRNA-induced mutation <i>in planta</i>. PCR amplification was conducted using the primers CsPDS-5-P1 and CsPDS-3-P2, which flank the target site within the <i>CsPDS</i> gene (Table S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093806#pone.0093806.s001" target="_blank">File S1</a>). Lanes 1-3, the template genomic DNA was digested with <i>Mfe</i>I. Lane 4, nondigested genomic DNA was used as a template. The PCR product in lane 1 resulted from Cas9/sgRNA-induced disruption of the <i>Mfe</i>I site and was therefore cloned into the PCR-BluntII-TOPO vector (Life Technologies) for sequencing. (c) Targeted mutations induced by Cas9/sgRNA in the <i>CsPDS</i> gene in sweet orange. Sequences of mutant variants of the <i>CsPDS</i> gene obtained from the clones constructed using the PCR product from lane 1 in Fig. 2b were aligned with the wild type sequence (top). The sgRNA-targeted <i>CsPDS</i> sequence is shown in red, and the mutations are shown in purple. (d) Measurement of the mutation rate of the <i>CsPDS</i> gene induced by Cas9/sgRNA. Genomic DNA was extracted from three samples (co-expression of Cas9 and sgRNA; expression of Cas9 alone; no expression of Cas9 and sgRNA), and subjected to PCR amplification using the primers CsPDS-5-P1 and CsPDS-3-P2. The PCR products were digested with <i>Mfe</i>I and analyzed by DNA gel electrophoresis (Lane 1, co-expression of Cas9 and sgRNA; Lane 2, expression of Cas9 alone; Lane 3, no expression of Cas9 and sgRNA). The mutation rate was calculated by dividing the intensity of the uncut band by the intensity of all the bands in each lane.</p

Table3_Base Editors for Citrus Gene Editing.DOCX

Base editors, such as adenine base editors (ABE) and cytosine base editors (CBE), provide alternatives for precise genome editing without generating double-strand breaks (DSBs), thus avoiding the risk of genome instability and unpredictable outcomes caused by DNA repair. Precise gene editing mediated by base editors in citrus has not been reported. Here, we have successfully adapted the ABE to edit the TATA box in the promoter region of the canker susceptibility gene LOB1 from TATA to CACA in grapefruit (Citrus paradise) and sweet orange (Citrus sinensis). TATA-edited plants are resistant to the canker pathogen Xanthomonas citri subsp. citri (Xcc). In addition, CBE was successfully used to edit the acetolactate synthase (ALS) gene in citrus. ALS-edited plants were resistant to the herbicide chlorsulfuron. Two ALS-edited plants did not show green fluorescence although the starting construct for transformation contains a GFP expression cassette. The Cas9 gene was undetectable in the herbicide-resistant citrus plants. This indicates that the ALS edited plants are transgene-free, representing the first transgene-free gene-edited citrus using the CRISPR technology. In summary, we have successfully adapted the base editors for precise citrus gene editing. The CBE base editor has been used to generate transgene-free citrus via transient expression.</p

Data_Sheet_1_Highly Efficient Generation of Canker-Resistant Sweet Orange Enabled by an Improved CRISPR/Cas9 System.pdf

Sweet orange (Citrus sinensis) is the most economically important species for the citrus industry. However, it is susceptible to many diseases including citrus bacterial canker caused by Xanthomonas citri subsp. citri (Xcc) that triggers devastating effects on citrus production. Conventional breeding has not met the challenge to improve disease resistance of sweet orange due to the long juvenility and other limitations. CRISPR-mediated genome editing has shown promising potentials for genetic improvements of plants. Generation of biallelic/homozygous mutants remains difficult for sweet orange due to low transformation rate, existence of heterozygous alleles for target genes, and low biallelic editing efficacy using the CRISPR technology. Here, we report improvements in the CRISPR/Cas9 system for citrus gene editing. Based on the improvements we made previously [dicot codon optimized Cas9, tRNA for multiplexing, a modified sgRNA scaffold with high efficiency, citrus U6 (CsU6) to drive sgRNA expression], we further improved our CRISPR/Cas9 system by choosing superior promoters [Cestrum yellow leaf curling virus (CmYLCV) or Citrus sinensis ubiquitin (CsUbi) promoter] to drive Cas9 and optimizing culture temperature. This system was able to generate a biallelic mutation rate of up to 89% for Carrizo citrange and 79% for Hamlin sweet orange. Consequently, this system was used to generate canker-resistant Hamlin sweet orange by mutating the effector binding element (EBE) of canker susceptibility gene CsLOB1, which is required for causing canker symptoms by Xcc. Six biallelic Hamlin sweet orange mutant lines in the EBE were generated. The biallelic mutants are resistant to Xcc. Biallelic mutation of the EBE region abolishes the induction of CsLOB1 by Xcc. This study represents a significant improvement in sweet orange gene editing efficacy and generating disease-resistant varieties via CRISPR-mediated genome editing. This improvement in citrus genome editing makes genetic studies and manipulations of sweet orange more feasible.</p

Average test set classification accuracy on <i>Caltech-UCSD Birds</i>.

Average test set classification accuracy on Caltech-UCSD Birds.</p

Table1_Base Editors for Citrus Gene Editing.DOCX

Base editors, such as adenine base editors (ABE) and cytosine base editors (CBE), provide alternatives for precise genome editing without generating double-strand breaks (DSBs), thus avoiding the risk of genome instability and unpredictable outcomes caused by DNA repair. Precise gene editing mediated by base editors in citrus has not been reported. Here, we have successfully adapted the ABE to edit the TATA box in the promoter region of the canker susceptibility gene LOB1 from TATA to CACA in grapefruit (Citrus paradise) and sweet orange (Citrus sinensis). TATA-edited plants are resistant to the canker pathogen Xanthomonas citri subsp. citri (Xcc). In addition, CBE was successfully used to edit the acetolactate synthase (ALS) gene in citrus. ALS-edited plants were resistant to the herbicide chlorsulfuron. Two ALS-edited plants did not show green fluorescence although the starting construct for transformation contains a GFP expression cassette. The Cas9 gene was undetectable in the herbicide-resistant citrus plants. This indicates that the ALS edited plants are transgene-free, representing the first transgene-free gene-edited citrus using the CRISPR technology. In summary, we have successfully adapted the base editors for precise citrus gene editing. The CBE base editor has been used to generate transgene-free citrus via transient expression.</p

Table2_Base Editors for Citrus Gene Editing.XLSX

Base editors, such as adenine base editors (ABE) and cytosine base editors (CBE), provide alternatives for precise genome editing without generating double-strand breaks (DSBs), thus avoiding the risk of genome instability and unpredictable outcomes caused by DNA repair. Precise gene editing mediated by base editors in citrus has not been reported. Here, we have successfully adapted the ABE to edit the TATA box in the promoter region of the canker susceptibility gene LOB1 from TATA to CACA in grapefruit (Citrus paradise) and sweet orange (Citrus sinensis). TATA-edited plants are resistant to the canker pathogen Xanthomonas citri subsp. citri (Xcc). In addition, CBE was successfully used to edit the acetolactate synthase (ALS) gene in citrus. ALS-edited plants were resistant to the herbicide chlorsulfuron. Two ALS-edited plants did not show green fluorescence although the starting construct for transformation contains a GFP expression cassette. The Cas9 gene was undetectable in the herbicide-resistant citrus plants. This indicates that the ALS edited plants are transgene-free, representing the first transgene-free gene-edited citrus using the CRISPR technology. In summary, we have successfully adapted the base editors for precise citrus gene editing. The CBE base editor has been used to generate transgene-free citrus via transient expression.</p

Average test set classification accuracy on different loss function.

Average test set classification accuracy on different loss function.</p

Accuracies sorted in descending order of each model on <i>mini</i>imageNet.

Accuracies sorted in descending order of each model on miniimageNet.</p

The channel-spatial attention network (C-SAM network) for 5 way-1 shot problem.

The channel-spatial attention network (C-SAM network) for 5 way-1 shot problem.</p