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

Abstract

Citrus canker disease affects citrus production. This disease is caused by Xanthomonas citri subsp. citri (Xcc). Previous studies confirmed that during Xcc infection, PthA4, a transcriptional activator like effector (TALE), is translocated from the pathogen to host plant cells. PthA4 binds to the effector binding elements (EBEs) in the promoter region of canker susceptibility gene LOB1 (EBE_PthA4-LOBP) to activate its expression and subsequently cause canker symptoms. Previously, the Cas12a/CBE co-editing method was employed to disrupt EBE_PthA4-LOBP of pummelo, which is highly homozygous. However, most commercial citrus cultivars are heterozygous hybrids and more difficult to generate homozygous/biallelic mutants. Here, we employed Cas12a/CBE co-editing method to edit EBE_PthA4-LOBP of Hamlin (Citrus sinensis), a commercial heterozygous hybrid citrus cultivar grown worldwide. Binary vector GFP-p1380N-ttLbCas12a:LOBP1-mPBE:ALS2:ALS1 was constructed and shown to be functional via Xcc-facilitated agroinfiltration in Hamlin leaves. This construct allows the selection of transgene-free regenerants via GFP, edits ALS to generate chlorsulfuron-resistant regenerants as a selection marker for genome editing resulting from transient expression of the T-DNA via nCas9-mPBE:ALS2:ALS1, and edits gene(s) of interest (i.e., EBE_PthA4-LOBP in this study) through ttLbCas12a, thus creating transgene-free citrus. Totally, 77 plantlets were produced. Among them, 8 plantlets were transgenic plants (#Ham_GFP1 - #Ham_GFP8), 4 plantlets were transgene-free (#Ham_NoGFP1 - #Ham_NoGFP4), and the rest were wild type. Among 4 transgene-free plantlets, three lines (#Ham_NoGFP1, #Ham_NoGFP2 and #Ham_NoGFP3) contained biallelic mutations in EBE_pthA4, and one line (#Ham_NoGFP4) had homozygous mutations in EBE_pthA4. We achieved 5.2% transgene-free homozygous/biallelic mutation efficiency for EBE_PthA4-LOBP in C. sinensis cv. Hamlin, compared to 1.9% mutation efficiency for pummelo in a previous study. Importantly, the four transgene-free plantlets and 3 transgenic plantlets that survived were resistant against citrus canker. Taken together, Cas12a/CBE co-editing method has been successfully used to generate transgene-free canker-resistant C. sinensis cv. Hamlin in the T0 generation via biallelic/homozygous editing of EBE_pthA4 of the canker susceptibility gene LOB1.

Keywords: CRISPR; Cas12a; Citrus; Xanthomonas; citrus canker; transgene-free genome editing.

Figure 1

Schematic representation of the binary vector GFP-p1380N-ttLbCas12a:LOBP1-mPBE:ALS2:ALS1 and its functional test. (A) Schematic diagram of GFP-p1380N-ttLbCas12a:LOBP1-mPBE:ALS2:ALS1. LOBP: the promoter region of LOB1. LB and RB, the left and right borders of the T-DNA region; CsVMV, the cassava vein mosaic virus promoter; GFP, green fluorescent protein; 35T, the cauliflower mosaic virus 35S terminator; CmYLCV, the cestrum yellow leaf curling virus promoter; NosP and NosT, the nopaline synthase gene promoter and its terminator; ttLbCas12a, temperature-tolerant LbCas12a containing the single mutation D156R; AtU6-26, Arabidopsis U6-26 promoter; target1, the 23 nucleotides of Type II LOBP highlighted by blue, was located downstream of protospacer-adjacent motif (PAM); HH, the coding sequence of hammerhead ribozyme; HDV, the coding sequence of hepatitis delta virus ribozyme; nCas9-mPBE, a plant codon-optimized base editor composed of rat cytidine deaminase APOBEC1, Cas9-D10A nickase (nCas9) and uracil glycosylase inhibitor (UGI); AtU6-26, Arabidopsis U6-26 promoter; target2 and target3, the 20 nucleotides of two CsALS alleles highlighted by blue, were located upstream of protospacer-adjacent motif (PAM); NptII, the coding sequence of neomycin phosphotransferase II. (B) Xcc-facilitated agroinfiltration of the binary vector GFP-p1380N-ttLbCas12a:LOBP1-mPBE:ALS2:ALS1. Xcc-pre-treated Hamlin leaf was agroinfiltrated with Agrobacterium cells harboring vector GFP-p1380N-ttLbCas12a:LOBP1-mPBE:ALS2:ALS1. After four days, GFP fluorescence was observed and photographed. A negative control was Agrobacterium cells harboring p1380-AtHSP70BP-GUSin. ttLbCas12a-directed LOBP indels and mPBE-mediated CsALS base editing were analyzed through Sanger sequencing. Among 200 colonies sequenced, there were expected mutations for LOBP and CsALS. x inside the parentheses indicates number of Sanger sequencing. The targeted sequence within LOBP and CsALS was underlined by black lines, and the mutant site was pointed out with arrows and highlighted by purple. Type I LOBP has one more G than Type II LOBP downstream of EBE_PthA4, and the G was highlighted by a black rectangle. The nucleotides different between two alleles of CsALS were highlighted by blue rectangles.

Figure 2

GFP detection and PCR verification of GFP-p1380N-ttLbCas12a:LOBP1-mPBE:ALS2:ALS1-transformed and transgene-free genome-edited Hamlin plants. (A) GFP fluorescence was observed in transgenic Hamlin plants, whereas wild type and transgene-free genome-edited plants did not show GFP. (B) Using a pair of primers Npt-Seq-5 and 35T-3PCR, wild type, transgenic and transgene-free Hamlin plants were analyzed. The wild type Hamlin and plasmid GFP-p1380N-ttLbCas12a:LOBP1-mPBE:ALS2:ALS1 were used as controls. M, 1kb DNA ladder.

Figure 3

Detection of genome editing of GFP-p1380N-ttLbCas12a:LOBP1-mPBE:ALS2:ALS1-transformed Hamlin by direct sequencing of CsALS PCR products (A) and LOBP PCR products (B). (A) The chromatograms of direct sequencing of CsALS PCR products. Primers CsALSP1 and CsALSP2 were used to amplify CsALS from wild type and transgenic Hamlin. Direct sequencing primer was CsALSP3. The base editing sites were shown by arrows. (B) The chromatograms of direct sequencing of LOBP PCR products. Primers LOB21 and LOB22 were used to amplify LOBP from wild type and transgenic Hamlin. Direct sequencing primer was LOB4. The mutation site or the beginning sites of multiple peaks were shown by arrows. The targeted sequence was underlined by black lines and EBE_PthA4-LOBP was highlighted by red rectangles.

Figure 4

Detection of genome editing of no-GFP-expressing Hamlin by direct sequencing of CsALS PCR products (A) and LOBP PCR products (B). (A) The chromatograms of direct sequencing of CsALS PCR products. Primers CsALSP1 and CsALSP2 were used to amplify CsALS from wild type and transgenic Hamlin. Direct sequencing primer was CsALSP3. The base editing sites were shown by arrows. (B) The chromatograms of direct sequencing of LOBP PCR products. Primers LOB21 and LOB22 were used to amplify LOBP from wild type and transgenic Hamlin. Direct sequencing primer was LOB4. The mutation site or the beginning sites of double peaks were shown by arrows. The targeted sequence was underlined by black lines and EBE_PthA4-LOBP was highlighted by red rectangles.

Figure 5

Sanger analysis of #Ham_NoGFP1 (A, B) and #Ham_NoGFP2 (c, d). Sanger sequencing results of #Ham_NoGFP1 and #Ham_NoGFP2. (A) As for CsALS of #Ham_NoGFP1, Type I allele contained 6^th, 7^th, 8^th C->T changes, and Type II allele was 6^th, 7^th C->T mutant among 10 colonies sequenced. (B) As for EBE_PthA4-LOBP of #Ham_NoGFP2, Type I allele had CCTTTTG deletion, and Type II allele had CCCTTTTG deletion. (C) As for CsALS of #Ham_NoGFP2, wild type and mutants were present among 10 colonies sequenced. (D) As for EBE_PthA4-LOBP of #Ham_NoGFP2, five of them are CCCTTTTGCCTTGAACTT deletion from Type I allele, and five of them are TTTTGCCTTAAC deletion from Type II allele.

Figure 6

Sanger analysis of #Ham_NoGFP3 (A, B) and #Ham_NoGFP4 (C, D). Sanger sequencing results of #Ham_NoGFP3 and #Ham_NoGFP4. (A) As for CsALS of #Ham_NoGFP3, Type I allele was 6^th, 7^th C->T mutant, and Type II allele had 6^th, 7^th, 8^th C->T mutation among 10 colonies sequenced. (B) As for EBE_PthA4-LOBP of #Ham_NoGFP3, Type I allele harbored CTTTTG deletion, and Type II allele contained CTTTtGCcttAAC deletion. (C) As for CsALS of #Ham_NoGFP4, Type I and Type II allele were 6^th, 7^th, 8^th C->T mutant among 10 colonies sequenced. (D) As for EBE_PthA4-LOBP of #Ham_NoGFP4, Type I and Type II allele had CCTTTTG deletion.

Figure 7

Canker-resistance in the transgenic and transgene-free EBE_PthA4-LOBP-edited Hamlin plants. Six days post Xcc inoculation, citrus canker symptoms were observed on wild type Hamlin, whereas no canker symptoms were observed on LOBP-edited Hamlin plants. As expected, XccpthA4:Tn5 (dCsLOB1.5) caused canker symptoms on all plants. dCsLOB1.5 induces LOB1 to cause canker symptoms by recognizing a different region from EBE_PthA4-LOBP.