Figure 2.

Agrobacterium-mediated CRISPR/Cas9 genome editing of NalaPDS in N. alata. (A) T-DNA constructs for Cas9-PF-NalaPDS. Cas9-PF-NalaPDS consists of a PF Cassette, which contains anthocyanin synthesis regulation gene PAP1 and early flower NtFT5 gene, as a visible marker, a Cas9 gene driven by 35S promoter, an sgRNA targeting the NalaPDS gene under the control of a U3 promoter, and a hygromycin resistance gene. The first exon of the N. alata PDS gene was selected as the target site for the gRNA. (B) Phenotypes of the pds mutants. Seedlings displayed albino phenotype are homozygous or bi-allele gene editing of NalaPDS. Scale bar=1 cm. (C) PCR product sequencing results of PDS mutant plants in (B) are aligned to the reference genome sequence. Different chromatograms of target position indicate different types of editing events, e.g., homozygous (Ho), bi-allele (Bi) and heterozygous (He) type editing. WT, wild-type; inserts and deletions are indicated as ‘i’ and ‘d’ respectively. (D) Total precise editing events categorized by different editing types, including homozygous (Homo), bi-allele (Bi-allel) and heterozygous (Heter) and wild-type (WT).