Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation

Abstract

Arabidopsis mutants produced by constitutive overexpression of the CRISPR/Cas9 genome editing system are usually mosaics in the T1 generation. In this study, we used egg cell-specific promoters to drive the expression of Cas9 and obtained non-mosaic T1 mutants for multiple target genes with high efficiency. Comparisons of 12 combinations of eight promoters and two terminators found that the efficiency of the egg cell-specific promoter-controlled CRISPR/Cas9 system depended on the presence of a suitable terminator, and the composite promoter generated by fusing two egg cell-specific promoters resulted in much higher efficiency of mutation in the T1 generation compared with the single promoters.

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

Fig. 2

Phenotypic segregation of T2 transgenic lines. Phenotypic segregation of T2 transgenic lines derived from two representative T1 lines with normal phenotypes. Seeds from 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 7 days. Hygromycin-resistant seedlings (T1) were transplanted to soil and allowed to grow for 20 days before photographing

Fig. 3

Structural and functional comparisons of twelve combinations of eight promoters and two terminators. a Seven combinations of EC1.1, EC1.2, or 2x35S promoters and rbcS E9 terminator (rbcS-E9t) or nos terminator (Nost). The pHEN2A-TRI and pHEN2C-TRI constructs have the same combination but different vector backbones: pGreen for the former and pCambia for the latter. The data for pHSN2A-TRI come from the publication and p2gR-TRI-A is renamed pHSN2A-TRI in this paper [26]. b Five combinations of five fusion promoters and the rbcS E9 terminator. Physical maps of the T-DNAs of seven (a) or five (b) CRISPR/Cas9 binary vectors are indicated. For each binary vector, the vector name, the promoter, the terminator, and the mutation frequencies of T1 transgenic plants are indicated at the same row under the maps. See Fig. 1 for RB/LB, zCas9, 2-sgRs, and Hyg. EC1p, EC1.1p or EC1.2p; 35Sen, CaMV 35S enhancer; EC1.2en, enhancer from EC1.2 promoter; LTM, likely triple mutant; Total, total number of T1 plants; Mosaics-I, type I mosaic plants with strong phenotypes indistinguishable from the double mutants; Mosaics-II, type II mosaic plants with the phenotypes appearing only in some parts of the whole plants. The ratios of T1 plants with the mutations (LTMs, Mosaics-I, or Mosaics-II) to total number of T1 plants are indicated