Hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) is the key enzyme in the biosynthetic pathway of chlorogenic acid. A homologous gene of NtHQT from Nicotiana tabacum was cloned and named NtHQT2. Amino acid sequence alignment and phylogenetic analysis revealed that NtHQT2 possessed the conserved HTLSD and DEFEG motifs of HQT protein and belonged to the same branch with HQTs from tobacco and other Solanaceae plants in terms of evolutionary relationships. By analyzing the expression patterns of NtHQT2 gene in different tissues of tobacco by qRT-PCR analysis, it was found that NtHQT2 gene was expressed in the root, stalk, leaf, axillary bud, and flower with higher expression levels observed in the leaves. CRISPR/Cas9 gene editing technology was used to perform targeted knockout of NtHQT2 gene, and three types of T0 generation gene-edited plants with NtHQT2 translation error were obtained. By quantifying the chlorogenic acid content in the leaves of homozygous T2 NtHQT2 gene-edited plants, it was observed that the edited plants exhibited a significant decrease in chlorogenic acid content compared to the control, indicating that NtHQT2 gene positively regulates the synthesis of chlorogenic acid.