Selection of the target gene and the sgRNA for CRISPR/Cas9

To examine whether the CRISPR/Cas9 system could be applied to I. nil, we selected the InDFR-B gene [accession number: AB006793]29 as the target of Cas9 endonuclease. Because null mutations in InDFR-B lead to anthocyanin-less stems, leaves and flowers, we visually distinguished the bi-allelic mutants during transformation. For the sgRNA sequence, we selected 20 bp in the fourth exon of the InDFR-B gene encoding a catalytic site of the DFR enzyme (Fig. 1a)33. The sgRNA sequence of InDFR-B shows high homology to those of InDFR-A and InDFR-C, with 19/20 and 18/20 matches in the nucleotide sequences, respectively, but only InDFR-B has the PAM next to the sgRNA sequence (Fig. 1a). Potential off-target sites of InDFR-B were searched with GGGenome (http://gggenome.dbcls.jp/) allowing no mismatch in seed sequence which is the most critical determinant of target specificity54, 55 and 3 bp mismatches in 20 bp sequence. Additional nine sites were derived as potential off-target sites (Supplemental Table S1).

Figure 1 CRISPR/Cas9-mediated targeted mutagenesis in InDFR-B. (a) Schematic representation of InDFR-A, -B, and -C target sequences. In InDFR-A and -C, the white letters in black highlight indicate mismatches with InDFR-B. In DFR-B, the 20-bp target-specific sequence is shown in blue highlight, and the PAM sequence (TGG) is shown in red highlight. SpeI restriction enzyme sites (ACTAGT) are underlined with green. The green triangles indicate the expected cleavage site of the CRISPR/Cas9 system. (b) T-DNA region of the all-in-one vector, pZD_AtU6gRNA_FFCas9 _NPTΙΙ. (c) Kanamycin-resistant regenerated shoots of plants transformed with the CRISPR/Cas9 system. Without targeted mutations, stems were coloured violet (left), whereas bi-allelically mutated stems remained green (right). (d) CAPS analysis of the target region in the InDFR-B locus. Total DNA was extracted from the leaves of transgenic plants and amplified by PCR. The PCR products were digested with the SpeI restriction enzyme, except -RE. M: marker (1,000, 700, 500, 200 and 100 bp); NT-RE: PCR product of a non-transgenic (NT) plant without restriction enzyme digestion. Numerals after # indicate independent T1 plants. N, B, L1, and L2 represent the phenotype of each plant. N: Violet stem and violet flower (same as NT); B: Green stem and white flower; L2: Green stem and violet flower; L1: Violet stem and pale-violet flower. (e) Sequences of targeted mutations in the InDFR-B locus. The NT type sequence is shown at the top and is designated (a). Deleted nucleotides are shown in dashes with black highlight (-). The inserted cytidine residue is shown with black highlight. NT sequences were detected using CAPS analysis (*) in chimaeric plants (#8-1 and 36-2). Full size image

Construct for the CRISPR/Cas9 system and transformation

An Arabidopsis codon-optimized Streptococcus pyogenes Cas9 expression cassette34, an sgRNA expression cassette, and a selective marker (neomycin phosphotransferase II: NPTII) were combined into a single plant binary vector to form an all-in-one vector for plant transformation (Fig. 1b). Cas9 was driven by the constitutive Ubiquitin4-2 promoter from Petroselinum crispum (pPcUbi)34, 35. The AtU6 promoter was used to express the sgRNA36, 37. The construct was introduced to secondary embryos to make stable transgenic I. nil using the Rhizobium (Agrobacterium) method7, 8. A total of 32 transgenic plants (T1) showing kanamycin resistance with the T-DNA insertion in the genome were obtained.

Identification of the mutants by appearance, CAPS analysis and DNA sequencing

Initially, visual screening based on the appearance of pigmentation with anthocyanin was performed. Non-transgenic cv. Violet plants exhibited violet-coloured stems; however, more than one-third of the regenerated plants showed anthocyanin-less stems, typical of the phenotype of null mutations in InDFR-B (Fig. 1c). Notably, we never observed a shift in colouring, namely, violet to green/green to violet within a single plantlet and its stems during growth.

Subsequently, we conducted a cleaved amplified polymorphic sequence (CAPS) analysis to detect mutations in the target region. We designed the expected cleavage site of CRISPR/Cas9 overlapping with the recognition sequence of the restriction enzyme SpeI (Fig. 1a). If the CRISPR/Cas9 cleaved target sequence and NHEJ occurred, then this restriction site would collapse, and the PCR-amplified DNA fragment would not be digested using SpeI. To detect mutations, total DNA was extracted from the transgenic leaves and PCR-amplified, and the resulting PCR products were subjected to SpeI digestion and analysed via agarose gel electrophoresis. The non-mutated PCR fragment of InDFR-B is 537 bp in length, and SpeI digestion produced 303- and 234-bp fragments; however, the mutated PCR fragments were not digested (Fig. 1d). Ten kanamycin-resistant transgenic plants showed SpeI-resistant single DNA bands, indicating that these plants are candidates of the bi-allelic mutants. Seventeen plants had both undigested and digested bands (Fig. 1d). Among these mutants, the #36-2 plants showed a shorter DNA fragment approximately 400 bp in length prior to SpeI digestion, suggesting a larger deletion in the locus (Fig. 1d). We also analysed the two orthologous loci, namely, InDFR-A and InDFR-C, using CAPS analysis and detected no mutations (Supplemental Fig. S1).

Moreover, the DNA sequences of the mutated sites in InDFR-B were determined. The error-free PCR-amplified fragments of the mono-allelic mutants, #8-1 and 36-2, and the bi-allelic mutants, #9-1 and 39-1, were cloned and subjected to DNA sequencing. All plants showed mutations in the predicted cleavage site (Fig. 1e). The detected insertion was one bp, whereas the deletions ranged from two to 98 bp. Interestingly, the L2 chimaeric plant #8-1 showed three patterns of DNA sequences in InDFR-B (two different mutations and one non-mutation), despite the diploid genome. The L1 chimaeric plant #36-2 had the longest deletion (98 bp), as predicted in the CAPS analysis (Fig. 1d,e and Supplemental Fig. S2). Moreover, we analysed all the nine off-target candidates sites having a PAM site listed in Supplemental Table S1 with DNA sequencing in the biallelic mutants #9-1 and 39-1, and found there was no mutation in the candidate sites (Supplemental Fig. S6).

Phenotype and genotype of the targeted mutagenesis

Bi-allelic mutants lost anthocyanin, resulting in green stems and white flowers (Fig. 2a). Non-mutated plants showed violet stems and flowers, similar to non-transgenic plants (NT) (Fig. 2b). Most of the mono-allelic mutants also showed violet stems and flowers, similar to non-mutated plants. Notably, plants #8-1 and #36-2, considered mono-allelic mutants based on CAPS analysis, showed green stems with violet flowers and violet stems with pale flowers, respectively (Fig. 2c,d). These two plants were considered periclinal chimaeras, as these phenotypes were the same as periclinal chimaeras resulting from transposon mutagenesis30. In I. nil, the L1 layer is responsible for most of the flower colour, whereas the L2 layer determines the stem colour and the genotype of gamete cells30. Therefore, plant #8-1 was considered an L2 chimaeric plant with a targeted mutation in DFR-B in the L2 layer, and plant #36-2 was considered an L1 chimaeric plant with a targeted mutation in DFR-B in the L1 layer. Because the L2 layer slightly contributed to the petal colour, the flowers of plant #36-2 were pale violet, as previously reported30. Then we checked the indel mutation and T-DNA existence at the root, stem, leaf and petal of #36-2. The root tissue does not contain L1 and L2 layers, because L3 forms the central tissues including the pith and roots56. The root of #36-2 had no mutation and no T-DNA insertion as expected (Supplemental Fig. S5). Therefore, we concluded that plant #36-2 was an L1 periclinal chimaera.

Figure 2 Flowers of CRISPR/Cas9-mediated dfr-b mutants. The appearances of flowers (top) and stems (middle) and a schematic drawing of the meristem layers and their functions (bottom). L1: epidermal layer; L2: sub-epidermal layer; L3: internal tissues. (a) A flower and stem of #9-1, a bi-allelic-mutant plant. (b) A flower and stem of I. nil cv. Violet, an NT plant. (c) A flower and stem of #8-1, an L2 periclinal chimaera plant and a representation of the meristem layers of the L2 chimaera showing bi-allelic mutation only in the L1 layer. (d) The flower and stem of #36-2, an L1 periclinal chimaera plant and a representation of the meristem layers of the L1 chimaera showing bi-allelic mutation only in the L2 layer. Full size image

We observed few somaclonal changes in anthocyanin pigmentation during cultivation. Periclinal chimaeric plants #8-1 and #36-2, considered L2 and L1 chimaeras, respectively, bore sectorial flowers only once during each life time, baring more than 30 flowers in total (Fig. 3a,b). Sectors of chimaeric plants were extensively studied using transposon mutants in the DFR-B locus in I. nil 30, and two potential causes were considered: somaclonal mutations and invasion of cells from an adjacent layer. We examined the total DNA extracted from sector cells using CAPS analysis and confirmed that these white sectors consisted of bi-allelically mutated cells (Fig. 3c).

Figure 3 Phenotypes and genotypes of sectorial chimaeric flowers. (a) A sectorial flower of the L2 chimaeric plant #8-1. (b) A sectorial flower of the L1 chimaeric plant #36-2. (c) CAPS analysis of the InDFR-B loci in sectorial chimaeric flowers shown in (a) and (b). Total DNA was extracted from the sectorial white tissues of the petals indicated by arrows (W) and other coloured tissues of the petal (C), which were subsequently used for PCR amplification. The PCR products were then digested using the SpeI restriction enzyme (+). -: without SpeI restriction enzyme digestion; M: marker (1,000, 700, 500, 200 and 100 bp); NT: Total DNA of an NT plant. Full size image

Inheritance of CRISPR/Cas9 system-induced mutations in subsequent generations

We observed the progeny (T2) of the transformed plants, and all results were essentially as expected. For example, the progeny of plant #9-1, bi-allelic mutants, showed green stems (24 plants), indicating that plant #9-1 (T1) was a perfect bi-allelic-targeted mutant of InDFR-B. Among the offspring of #9-1, targeted mutants without T-DNA insertions were observed, suggesting that the targeted mutations at InDFR-B and T-DNA were not co-inherited in #9-1. These plants are targeted mutants and are considered transgenic plants based on process-based definitions and non-transgenic plants based on product-based definitions38, 39.

We also observed the progeny (T2) of the L2 chimaeric mutant #8-1 and the L1 chimaeric mutant #36-2. The progeny of plant #8-1 showed green stems (16 plants), and the progeny of plant #36-2 showed violet stems (50 plants). We examined the T-DNA retention of the 24 progenies of the L1 chimaeric mutant #36-2 using PCR amplification to detect a fragment of the NPTΙΙ gene. None of Supplemental Fig. S3 the T2 plants of #36-2 progeny had T-DNA insertions in their genomic DNA (). Because gamete cells originated from L2 cells30, these results reconfirmed that plant #8-1 was an L2 chimaeric plant and that plant #36-2 was an L1 chimaeric plant.