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Background: Amber-like compounds form in tobacco (Nicotiana tabacum) during leaf curing and impact aromatic quality. In particular, cis-abienol, a polycyclic labdane-related diterpenoid, is of research interest as a precursor of these compounds. Glandular trichome cells specifically express copalyl diphosphate synthase (NtCPS2) at high levels in tobacco, which, together with NtABS, are major regulators of cis-abienol biosynthesis in tobacco. Results: To identify the genes involved in the biosynthesis of cis-abienol in tobacco, we constructed transgenic tobacco lines based on an NtCPS2 gene-knockdown model using CRISPR/Cas9 genome-editing technology to inhibit NtCPS2 function in vitro. In mutant plants, cis-abienol and labdene diol contents decreased, whereas the gibberellin and abscisic acid (ABA) contents increased compared with those in wild-type tobacco plants. RNA sequencing analysis revealed the presence of 9514 differentially expressed genes (DEGs; 4279 upregulated, 5235 downregulated) when the leaves of wild-type and NtCPS2-knockdown tobacco plants were screened. Among these DEGs, the genes encoding cis-abienol synthase, ent-kaurene oxidase, auxin/ABA-related proteins, and transcription factors were found to be involved in various biological and physiochemical processes, including diterpenoid biosynthesis, plant hormone signal transduction, and plant-pathogen interactions. Conclusions: The present study provides insight into the unique transcriptome profile of NtCPS2 knockdown tobacco, allowing for a better understanding of the biosynthesis of cis-abienol in tobacco. [ABSTRACT FROM AUTHOR]