Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory

Abstract

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated with protein CAS9) is a genome-editing tool that has been extensively used in the last five years because of its novelty, affordability, and feasibility. This technology has been developed in many plant species for gene function analysis and crop improvement but has never been used in chicory (Cichorium intybus L.). In this study, we successfully applied CRISPR/Cas9-mediated targeted mutagenesis to chicory using Agrobacterium rhizogenes-mediated transformation and protoplast transfection methods. A U6 promoter (CiU6-1p) among eight predicted U6 promoters in chicory was selected to drive sgRNA expression. A binary vector designed to induce targeted mutations in the fifth exon of the chicory phytoene desaturase gene (CiPDS) was then constructed and used to transform chicory. The mutation frequency was 4.5% with the protoplast transient expression system and 31.25% with A. rhizogenes-mediated stable transformation. Biallelic mutations were detected in all the mutant plants. The use of A. rhizogenes-mediated transformation seems preferable as the regeneration of plants is faster and the mutation frequency was shown to be higher. With both transformation methods, foreign DNA was integrated in the plant genome. Hence, selection of vector (transgene)-free segregants is required. Our results showed that genome editing with CRISPR/Cas9 system can be efficiently used with chicory, which should facilitate and accelerate genetic improvement and functional biology.

Keywords: Agrobacterium rhizogenes-mediated transformation; CRISPR/Cas9; Cichorium intybus; multiplex genome editing; phytoene desaturase; protoplast transformation.

Figure 1

Chicory U6 and Arabidopsis U6-26 gene sequence partial alignment. The highly conserved parts of the sequences are in black while the Upstream Sequence Elements (USE), the TATA-boxes and the U6 small nuclear (snRNA) genes are marked with black bars. The transcription start sites are indicated (red frame).

Figure 2

Overview of the experimental design for CiPDS disruption. (A) Schematic position of the two guide RNAs (red boxes) targeting the fifth exon of CiPDS. The blue boxes indicate exons; the black lines indicate introns. (B) Schematic view of pKanCiU6-1p-sgRNAscaffold. Between the CiU6-1p and the sgRNA scaffold (scaff), a ccdB gene driven by the LacZ promoter (counter selection marker) is surrounded by BbsI (second-generation enzyme) recognition sites, which allows the ligation of the hybridized guide adaptor shown in C. Red B-L depicts the orientation of the BbsI recognition site which achieves the cleavage on the left side. Blue B-R depicts the orientation of the BbsI recognition site which achieves cleavage on the right side. (C) Sequences of the complementary guide adaptors with the 19 pb guide sequence (yellow), the transcription initiator nucleotide G (bold), and the binding sites necessary to insert the hybridized guide adaptor into the pKanCiU6-1p-sgRNAscaffold (red). (D) Representation of the vector resulting from the insertion of the hybridized guide adaptors in the pKanCiU6-1p-sgRNAscaffold, (note that one vector is constructed for each guide). The primers used for preparation of CiU6-1p-guide-sgRNA cassettes for Golden Gate Cloning are also shown. (E) Representation of the final plasmid pYLCRISPR-sgRNA1-sgRNA2 resulting from the cloning of the two cassettes into the pYLCRISPR/Cas9P_35S-B [36]. RB = Right Border, LB = Left Border, NLS = Nuclear Localization Signal.

Figure 3

Phenotype of genome edited plants. (A) Wild-type (WT) shoot emerging from a WT callus. (B) CiPDS edited albino shoots emerging from a callus. (C) Hairy root line transformed with wild-type A. rhizogenes strain 15834 with emerging shoot. (D) Albino shoot emerging from hairy root line engineered to knock out CiPDS.

Figure 4

Sequence analysis of the two alleles of the 10 albino hairy root lines. The target sequences are depicted in blue and the PAM sequences in red. The change in the number of nucleotides is shown on the right of each allele sequence. A + indicates an insertion and a – stands for a deletion. WT indicates that there is no mutation. Example: -5/WT = deletion of 5 nucleotides on the first target site, no mutation on the second target site. For the L5: (R) = Hairy root stage sequence, (P) = plant stage sequence. The length of the amino acid chain in the truncated protein is shown on the right. Lx.x: line x allele x; AA: Amino Acid chain length; T1: Target1; T2: Target2.

Figure 5

Sequence analysis of the two alleles of 9 albino calli obtained after protoplast transformation. The target sequences are depicted in blue and the PAM sequences in red. The change in the number of nucleotides is shown on the right of each allele sequence. A + or a – indicate an insertion or a deletion, respectively. A S means that there is a substitution. WT indicates that there is no mutation. Example: -5/WT = deletion of 5 nucleotides on the first target site, no mutation on the second target site. The length of the amino acid chain in the truncated protein is shown on the right. Cx.x: Callus x allele x; AA: Amino Acid chain length; T1: Target1; T2: Target2. (+ npb) = n base pairs resulting from insertion of plasmid parts.