Fig 1.
Generation of a FT expression cassette containing pHEE CRISPR/Cas9 construct for AITR1 editing.
(A) pHEE vector with a FT expression cassette. The full-length ORF sequence of GmFT2a was synthesized, and cloned into pUC19 vector under the control of the 35S promoter and terminated by the nos sequence. The 35S:GmFT2a-nos cassette was amplified by PCR and then inserted into the pHEE vector at the Pme1 site by using Gibson assembly to generate pHEE-FT vector. (B) The sgRNA expression cassettes in the pHEE-FT-AITR1 construct. The sgRNA sequences corresponding to the target sequences of AITR1 were introduced into the sgRNA expression cassettes by PCR amplification, followed by Golden Gate reaction with the pHEE-FT vector. (C) Target sequences in AITR1. Numbers indicated the nucleotide position relative to the first nucleotide in the coding sequence of AITR1, PAM sites after the target sequences were indicated in the brackets.
Fig 2.
Early bolting phenotypes observed in the T1 and T2 transgenic plants.
(A) Early bolting phenotype in some T1 transgenic plants. Transgenic plants were selected on antibiotic-containing 1/2 MS plates, and ~5-day-old transgenic seedlings were transferred into soil pots and grown in a growth room. As a control, seeds of Col wild type were germinated on 1/2 MS plates, and seedlings ~5-day-old were transferred into soil pots. Pictures were taken 10 days after the transfer. (B) Bolting time of the T1 transgenic plants. The date of bolting for the plants was recorded, and days after the transfer were calculated. For Col wild type plants, n = 11. For transgenic plants, n = 53. (C) Bolting time of the T2 transgenic plants from 2 independent T1 lines. T2 seeds were sown directly into soil pots and growth room. The date of bolting for the plants was recorded, and bolting time was calculated. Data represent mean ± SD of 9–40 plants.
Fig 3.
AITR1 editing status in T1 transgenic plants.
(A) PCR amplification of AITR1 coding sequence in the T1 transgenic plants. DNA was isolated from leaves collected from Col wild type and individual T1 transgenic plants, and PCR was used to amplify coding sequence of AITR1. Amplification of ACT2 was used as a control. Picture is image of PCR results for Col wild type and T1 transgenic plants lines 1 to 15, showing the 2 PCR product bands obtained in line 14, and 1 band for Col and other lines. M, 250 bp DNA maker. (B) AITR1 editing status in sequenced individual T1 transgenic plants. PCR products were recovered from gel and sequenced. Sequencing results were examined and aligned with coding sequence of AITR1 to check the editing status in the T1 transgenic plants. Early bolting lines were numbered sequentially, whereas lines normal bolting plants were numbered according to their initial location in the tray with a letter and a number. wt, not edited, -/+, edited but heterozygous, ins, homozygous or biallelic editing with nucleotide insertions as indicated, N/A, not sequenced.
Fig 4.
Phenotype segregation in the T2 generation of selected transgenic lines.
(A) Bolting phenotype of the T2 plants from a single T1 transgenic line. T2 seeds were collected from selected T1 transgenic lines and sown directly into soil pots and grown in a growth room. Col wild type plants were generated and grown side by side with the T2 plants as a control. Pictures were taken 17 days after germination. Arrows indicate plants that did not bolt early. (B) Bolting phenotype segregation of the T2 plants from 4 T1 transgenic lines. Chi square analysis was performed on omni calculator (https://www.omnicalculator.com/statistics/chi-square).
Fig 5.
T2 plants with normal bolting time are transgene-free plants.
DNA was isolated from leaves collected from 4–6 individual normal bolting T2 plants for each of the 4 lines, and PCR was used to amplify Cas9 fragment. For each line, DNA was also isolated from two early bolting plants, and used a positive control for Cas9 amplification. PCR amplification of ACT2 was used as a control. M, 250 bp DNA maker.
Fig 6.
Isolation of genome edited transgene-free aitr1 mutants.
Transgene-free aitr1 mutants isolated from line 14 (A) and lines 6, 9 and 15 (B). DNA was isolated from leaves collected from at least 2 individual normal bolting T2, and used as a template to amplify the coding sequence of AITR1 and Cas9. Amplification of ACT2 was used as a control. The PCR products were recovered from gel and sequenced. M, 250 bp DNA maker. Sequencing results were compared with coding sequence of AITR1 to check the editing status. ORF of the AITR1 sequences in the aitr1 mutants were identified on ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/), and corresponding amino acid sequences were used for alignment with AITR1 amino acid sequences. Triangle indicates fragment deletion, arrow indicates nucleotide insertion, and underlines in the sequence results indicate the PAM sites. Numbers in the alignment indicate the position of amino acid relative to the first Met of AITR1.
Fig 7.
Simplified procedure for generating genome edited tansgene-free mutants by using FT expression cassette-containing CRISPR/Cas9 construct.
Plants can be transformed and transgenic plants can be selected in a way similar to that for other constructs. Within the T1 transgenic plants, select plants with early-bolting phenotype, and sequence to examine genome editing status. Keep only genome edited plants. In the T2 plants germinated from seeds of the individual T1 plants, keep only those that bolt normally, and sequence to identify genome edited plants. These plants are genome edited transgene-free mutants. If necessary, confirm the transgene-free status by PCR amplification of Cas9 fragment.