The CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat-associated protein 9) is a powerful genome-editing tool in animals, plants, and humans. This system has some advantages, such as a high on-target mutation rate (targeting efficiency), less cost, simplicity, and high-efficiency multiplex loci editing, over conventional genome editing tools, including meganucleases, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs). One of the crucial shortcomings of this system is unwanted mutations at off-target sites. We summarize and discuss different approaches, such as dCas9 and Cas9 paired nickase, to decrease the off-target effects in plants. According to studies, the most effective method to reduce unintended mutations is the use of ligand-dependent ribozymes called aptazymes. The single guide RNA (sgRNA)/ligand-dependent aptazyme strategy has helped researchers avoid unwanted mutations in human cells and can be used in plants as an alternative method to dramatically decrease the frequency of off-target mutations. We hope our concept provides a new, simple, and fast gene transformation and genome-editing approach, with advantages including reduced time and energy consumption, the avoidance of unwanted mutations, increased frequency of on-target changes, and no need for external forces or expensive equipment.

Populus trees meet continuous difficulties from the environment through their life cycle. To warrant their durability and generation, Populus trees exhibit various types of defenses, including the production of secondary metabolites. Syntheses derived from the shikimate-phenylpropanoid pathway are a varied and plentiful class of secondary metabolites manufactured in Populus. Amongst other main classes of secondary metabolites in Populus are fatty acid and terpenoid-derivatives. Many of the secondary metabolites made by Populus trees have been functionally described. Any others have been associated with particular ecological or biological processes, such as resistance against pests and microbial pathogens or acclimatization to abiotic stresses. Still, the functions of many Populus secondary metabolites are incompletely understood. Furthermore, many secondary metabolites have therapeutic effects, leading to more studies of secondary metabolites and their biosynthesis. This paper reviews the biosynthetic pathways and therapeutic impacts of secondary metabolites in Populus using a genomics approach. Compared with bacteria, fewer known pathways produce secondary metabolites in Populus despite P. trichocarpa having had its genome sequenced.

1-Deoxy-d-xylulose-5-phosphate synthase (DXS) is the rate-limiting enzyme in the plastidial methylerythritol phosphate pathway (MEP). In this study, PtDXS (XM_024607716.1) was isolated from Populus trichocarpa. A bioinformatics analysis revealed that PtDXS had high homology with the DXSs of other plant species. PtDXS expression differed among plant tissues and was highest in young leaves and lowest in roots. The recombinant protein was produced in Escherichia coli BL21 (DE3), purified, and its activity evaluated. The purified protein was capable of catalyzing the formation of 1-deoxy-d-xylulose-5-phosphate (DXP) from glyceraldehyde-3-phosphate and pyruvate. A functional color assay in E. coli harboring pAC-BETA indicated that PtDXS encodes a functional protein involved in the biosynthesis of isoprenoid precursors. The treatment of P. trichocarpa seedlings with 200 μM abscisic acid (ABA), 200 mM NaCl, 10% polyethylene glycol₆₀₀₀, and 2 mM H₂O₂ resulted in increased expression of PtDXS. The ABA and gibberellic acid contents of the transgenic lines (Poplar Nanlin 895) were higher than wild types, suggesting that DXS is important in terpenoid biosynthesis. Overexpression of PtDXS enhanced resistance to S. populiperda infection. Furthermore, the transgenic lines showed decreased feeding by Micromelalopha troglodyta, supporting the notion that PtDXS is a key enzyme in terpenoid biosynthesis.