Fig. 1 Homology comparison of NtREV1 and NtREV2 amino acid sequences

Fig. 2 Schematic structure and target site selection of NtREV and vector construction A: Schematic diagram of REV gene exons and introns, as well as the locations of the target editing sequences. B: Nucleic acid electrophoresis of double enzyme digestion of REV gene the target editing sequences

Fig. 3 Construction of the plant binary expression vector pCAMBIA1300-pYAO-Cas9-NtREV A: Nucleic acid electrophoresis of PCR amplification of pCAMBIA1300-pYAO-Cas9-NtREV. B: Nucleic acid electrophoresis of double enzyme digestion of pCAMBIA1300-pYAO-Cas9-NtREV

Fig. 4 Nucleic acid electrophoresis for the detection of transgenic plants A: Nucleic acid electrophoresis for detecting plant editing of the REV gene at target C15. B: Nucleic acid electrophoresis for detecting plant editing of the REV gene at target C16

Fig. 5 Mutation form analysis of NtREV edited by the CRISPR/Cas9 A: Mutation of REV target C16 sequence; B: mutation of REV target C15 sequence. Underscores indicate PAM sequences, blue words indicate replacement bases, red words indicate insertion bases, and - indicates missing bases

Fig. 6 Leaf morphological phenotypes of mutant and wild-type materials A-F: Leaf development morphology of the double-copy homologous mutants, including funnel-shaped leaves, lotus-shaped leaves and other morphologies; G-H: leaf development morphology of the single-copy homologous mutants; I: wild type growth state in the corresponding period; bar=1 cm

Fig. 7 Editing forms of Ko-C15Ntrev and Ko-C16Ntrev mutants in tobacco The double-copy homologous mutants of Ko-C15NtREV; Ko-C16NtREV editing form is the single-copy homologous mutants

Fig. 8 Observations of apical buds of mutant and wild-type ones A, B, C: Apical bud phenotypes of tobacco seedling wild-type, Ko-C16Ntrev and Ko-C15Ntrev mutant under stereomicroscope, respectively; D, E, F: apical meristem phenotypes of tobacco seedling wild-type,Ko-C16Ntrev and Ko-C15Ntrev mutant under scanning electron microscopy, respectively; G, H, I: tobacco seedling wild-type, Ko-C16Ntrev and Ko-C15Ntrev mutant corresponding to the above phenotypes, respectively

Fig. 9 Phenotypes of plant height, leaf numbers and fresh weight of C16Ntrev mutants A, B, C: Statistical analysis of plant height, leaf numbers and fresh weight content of axillary buds of WT and Ko-C16Ntrev mutants. D and E: The phenotype of lateral buds of WT and Ko-C16Ntrev mutants, respectively. Bar= 5 cm. The data represent mean ± SD, n ≥ 10; ** indicates significance P<0.01