CRISPR-Mediated Editing of LATE Gene

Editing of the LATE gene was performed using the CRISPR/Cas9 system. Guide RNA sequences were amplified with primer sets (Supplementary Table S1) and pGTR (Xie et al. 2015 (link)) as a template, and then the amplified fragments were cloned into the AarI site of the pKI1.1R vector (Tsutsui and Higashiyama 2017 (link)) using Gibson Assembly® Master Mix (New England BioLabs). Using this vector, Arabidopsis plants were transformed via Agrobacterium in accordance with the floral dip method. The T₁ generation of transgenic plants was screened on MS medium containing 30 mg l⁻¹ hygromycin and 250 mg l⁻¹ vancomycin, followed by transfer into the soil. Successful genome editing of the plants was confirmed by PCR and sequence analysis. Those individuals that were homozygous at the editing sites of the LATE gene and Cas9-free were selected from the T₃ progeny. These plants were grown at 22°C under long-day and short-day conditions. Flowering time was quantified by counting the total leaf number when the main inflorescence stem reached to about 5 cm and the number of days after sowing until flower bud formation is confirmed. Statistical analysis comparing WT and genome-edited plants was performed using Student’s t-test (p<0.05).