Fig. 3.

TvDAO helps to enrich CRISPR-edited alleles in Arabidopsis. A Diagram of the expression cassettes for dTvDAO, Cas9 (SpCas9), gRNAs and hygromycin resistance gene (HygR) in the binary plasmid. NLS, nuclear localization signal; E9_ter, E9 terminator. B Rationale of CRISPR-induced single-strand annealing (SSA) repair to restore a functional TvDAO selection marker. In the dead TvDAO (dTvDAO) reporter, the most 5′ (1–279) and 3′ segments (789–1068) of TvDAO were colored in green. The middle segment (280–788) of TvDAO was colored in blue and the two copies (LH and RH) of it were spacered by two consecutive stop codons and a Cas9 target site from the human hEfemp1 locus. C PCR-based genotyping confirms successful SSA repair of dTvDAO in d-serine-resistant transgenic T₁ plants. S, d-serine selection. H, hygromycin selection. Transgenic TvDAO #2 plant was used as a positive control. D Sanger sequencing of target amplicons validates the editing of all four target sites (dTvDAO, CPC, ETC2 and TRY) in the dTvDAO-gRNAs-Cas9 #S1 (Cas9 #S1) plant. Blue letters in dTvDAO represent the C-terminal coding sequences of LH and RH, while green letters represent the coding sequences adjacent to the C-terminus of RH. Black bold letters mark PAMs and target sequences of gRNAs are underlined. Deletions are indicated by red dashes, while insertions are colored in red. E The Cas9 #S1 plant exhibits visible clustering of trichomes on the leaf surface as indicated by red arrows. Scale bar = 1 cm. F Summary of mutation frequencies at individual target sites in transgenic T₁ plants selected by d-serine or hygromycin. HO, HE, Bi and WT denote homozygote, heterozygote, bi-allele and wild type, respectively