Fig. 1.

Generation of aitr23456 quintuple and aitr123456 sextuple mutants. a Alignment of the CRISPR/Cas9 target sequences of AITR3 and AITR4 in the Col wild type and the aitr23456 quintuple and aitr123456 sextuple mutants. The mutants were generated by transforming the aitr256 triple and aitr1256 quintuple T-DNA insertion mutant plants, respectively with a pHEE CRISPR/Cas9 construct that simultaneously targeting AITR3 and AITR4. Genome editing status in the selected T1 transgenic plants was examined by sequencing. Homozygous Cas9-free mutants were isolated from T2 or T3 progeny of the individual genome edited T1 plants. Numbers indicate the nucleotide position relative to the start condons of AITR3 and AITR4, respectively. Underlines indicate the PAM sites immediately after the target sites. b Amino acid sequence alignment of AITR3 and AITR4 in the Col wild type and the aitr23456 quintuple and aitr123456 sextuple mutants. The coding sequences of AITR3 and AITR4 in the aitr23456 quintuple and aitr123456 sextuple mutants were used for ORF analysis on ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/). Predicated amino acid sequences were used for alignment with wild type AITR3 and AITR4, respectively. Numbers indicate amino acid position relative to the first Met residue. Underlines indicate the fully or partial conserved LxLxL motif in AITRs