Targeted genome editing in Nicotiana tabacum using inducible CRISPR/Cas9 system

Targeted genome editing has been achieved in multiple plant species using the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) system, in which the Cas9 gene is usually driven by constitutive promoters. However, constitutive expression of Cas9 is not necessary and can be harmful to plant development. In this study, we developed an estrogen-inducible CRISPR/Cas9 system by taking advantage of the chimeric transcription activator XVE and tested the efficacy of this inducible system in Nicotiana tabacum by targeting the phytoene desaturase (NtPDS) gene, whose mutation resulted in albino phenotypes. Treatment of four independent transgenic lines with exogenous estradiol successfully induced targeted mutagenesis in NtPDS. Sanger sequencing assay uncovered the presence of indel mutations (nucleotides insertions or deletions) at the target site as expected, and at least two types of mutations were identified for each line. Transgenic plants with mutated NtPDS gene after estradiol treatment exhibited pale green or incomplete albino leaves. Moreover, the expression of Cas9 in transgenic plants was strongly induced by estradiol treatment. Our results demonstrate the efficacy of XVE-based CRISPR/Cas9 system in N. tabacum, and the system reported here promises to be a useful approach for conditional genome editing, which would facilitate the study of genes of interest, especially those developmentally important genes.