Fig. 1

Efficient genome editing in dicots using calreticulin promoter-driven CRISPR/Cas system. A Schematic illustration of CRISPR/Cas9 systems used in this study. NLS: nuclear location signal; eu: intronless tobacco extension terminator; 2A: self-cleaving 2A peptide. The bar in light blue: tRNA sequence. B Comparison of the ho/bi mutations based on the ratios of albino plantlets generated by targeting tobacco PDS genes with various CRISPR/Cas9 systems, the standard deviation (SD) is derived from three replicates. C Comparative analysis of mutation editing efficiencies in the tobacco T0 generation generated via pDC30, pDC40, and pDC45 in the NtCIPK, NtWOX1, NtBRC1, NtCEN, NtCLC and NtPPD target genes. D The ho/bi mutations of tobacco T0 seedlings induced by various CRISPR/Cas9 cassettes for the six targeted genes in this study. E Agarose gel electrophoresis and Sanger sequencing of fragment deletions generated by pDC45_PDSdsg. The target was NtPDS gene. The yellow arrowheads show the excised fragments used for sequencing. F Chromosomal segment deletion efficiency created by the pDC45_dsg system. The location of the chromosomal fragment deletion is referenced to the tobacco reference genome. G The phenotype of transgenic tobacco lines generated by various pDC constructs. Plates #1 and #2 display randomly selected explants from two individual experiments. Red arrowheads indicate the absolute albino shoots of NtPDS disruption seedlings, as well as the bladeless leaf phenotype produced by the targeted knockout of NtWOX1 allelic genes. “S1” and “S3” indicate different stages of tobacco regeneration. EV, pDC45 vector without a target sequence. H Diverse phenotypes of tobacco ho/bi T0 lines with pDC45_PDSsg + WOX1sg construct. The numbers highlighted in yellow and red show the count of ho/bi lines and Hyg-resistant lines, respectively. I Phylogenetic tree of CRT proteins based on amino acid sequences from different species. The sequence information employed for alignment is detailed in the supplementary materials. Monocots and dicots were indicated in different groups. A filled circle with red and blue was marked before tobacco PCE8 and other CRTs in Solanum species. The light green filled circle represents lettuce CRT protein. J Sequence alignment of CRTs in different plant species. Amino acid sequences were aligned using the DNAMAN alignment program. K Gene structure and target sites of lettuce LsPDS gene. L The phenotypes of T0 seedlings generated by pDC30 and pDC45 vector, The albino shoots indicate complete knockout of lettuce PDS gene. M Sanger sequencing of PCR product from LsPDS edited lines. Nucleotide sequences are indicated by yellow for the target and magenta for the PAM sequence, respectively. N Editing efficiency analysis of T0 transgenic lettuce harboring different editing systems. “All types” indicates that every form of editing events was included. “ho/bi type” is restricted to the enumeration of solely ho/bi mutations. O Observation of early bolting and fruit setting in T0 transgenic tomato and tobacco. ‘NT’ stands for non-transgenic lines while ‘#’ refers to individual edited lines. P Sequence chromatogram of PCR products of pDC45_Fast transgenic plants, the spacer and PAM sequence is underlined