Fig. 1

Arabidopsis T1 homozygous triple mutants obtained via EPC CRISPR/Cas9. a Physical maps of the T-DNAs of two CRISPR/Cas9 binary vectors, each harboring Cas9 driven by the egg-cell specific promoter EC1.2p and two sgRNA genes driven by Pol-III promoters U6-26p and U6-29p, respectively. RB/LB, T-DNA right/left border; EC1.2p, EC1.2 promoter; rbcS-E9t, rbcS E9 terminator; Nost, nos gene terminator; sgR, sgRNA; 2-sgRs, two sgRNA expression cassettes; zCas9, Zea mays codon-optimized Cas9; U6-26p and U6-29p, two Arabidopsis U6 gene promoter; U6-26t, U6-26 terminator with downstream sequence; Hyg, hygromycin-resistance gene. For the sgRNAs, the yellow part represents 20-bp target and the green part represents 76-bp sgRNA scaffold. b The alignment of the sgRNA with its target genes and potential off-targets. Only aligned regions of interest are displayed. rc, reverse complement. c Phenotypes of two triple mutants segregated from T1 transgenic lines. The other plants in the same pot are from the same batch of T1 transgenic lines with normal phenotypes. Seeds from the T0 plants were sown on MS medium containing 25 mg/L hygromycin, vernalized at 4 °C for 3 days, and grown under long-day conditions (16 h light/8 h dark) at 22 °C for 9 days. Hygromycin-resistant seedlings (T1) were transplanted to soil and allowed to grow for 33 days before photographing