CRISPR/Cas9 technology facilitates gene editing by constructing single guide RNA (sgRNA) that is both homologous and complementary to the target sequence, thereby achieving precise positioning and accurate cutting for gene knockout or knock-in. The CRISPR/Cas9 system has been applied to a variety of medicinal plants, including dicots, tobacco, dendrobium, soybean, and radish (Alagoz et al., 2016; Hong et al., 2021; Kui et al., 2016; Sugano et al., 2018; Wang et al., 2019). In the context of plant gene editing, CRISPR technology is primarily utilized to modify genes and regulate the expression of various enzymes involved in the synthesis pathways of medicinal active ingredients. Key enzyme regulation strategies include enzyme inhibition, branched chain blockade, and modular classification of secondary metabolic pathways to investigate the functions of essential enzymes. The enzyme inhibition strategy employs the CRISPR system to silence enzymes within the biosynthetic pathway, thereby altering the content of target secondary metabolites (Zhou et al., 2012). This approach is the most prevalent strategy for studying plant secondary metabolic pathways and has been utilized in numerous projects investigating functional genes in secondary metabolite production pathways, including those related to salvianolic acids, tanshinones, anthocyanins, triterpene saponins, and cocaine. Examples of these studies include modifications of CRISPR technology for multi-gene editing, such as the double knockout of Salvia miltiorrhiza genes SmDML3 and SmMET1 using CRISPR/Cas9 technology, which facilitates the rapid and efficient knockout of target genes or entire gene families (Xing et al., 2014). Furthermore, one study employed CRISPR multiplex knockdown technology to simultaneously knock out two XylT genes and four FucT genes (12 alleles in total) that regulate plant-specific glycan glycosylation in tobacco, resulting in a deficiency of glycoproteins (Mercx et al., 2017). In another study, researchers utilized the CRISPR system to target and knock out the NtPDS and NtPDR6 genes in tobacco (Gao J. et al., 2015), demonstrating that the CRISPR/Cas9 system is an effective tool for inducing targeted mutations within the tobacco genome. Similarly, the CRISPR/Cas9 system was employed to knock out β (1,2)-xylosyltransferase (OsXylT) and α (1,3)-fucosyltransferase (OsFucT) genes in rice, highlighting the versatility of CRISPR technology in plant genome editing (Jung et al., 2021).

Branched chain blocking strategies focus on obstructing or disrupting competing pathways within the synthesis pathway of the desired active substance, thereby diverting precursor substances to the target synthesis pathway. This method has been applied in the study of artemisinin and triterpene saponin metabolic pathways, specifically targeting the SQS and CYP716A53v2 genes, respectively. This strategy is relevant not only to plants but also to the investigation of microbial cell factories. Recent studies on the metabolism of pharmaceutical active ingredients have identified simple organisms, such as Escherichia coli and Saccharomyces cerevisiae, as the primary hosts for genetic engineering. These organisms have been modified through gene editing to alter their original metabolic pathways, enabling low-cost production of target active substances (Huang et al., 2014). Notably, this included the use of CRISPR to enhance MVA metabolic flux in Saccharomyces cerevisiae cells while blocking the sterol branch pathway, which resulted in a significant increase in the content of the precursor mevalonate in the mutant strain (Jakočiūnas et al., 2015). Additionally, the group is collaborating with Amyris to engineer bacteria for high artemisinic acid production, thereby boosting the production of this compound (Paddon et al., 2013; Ro et al., 2006). This technology has also been applied to modify S. cerevisiae cells to develop engineered yeast capable of simultaneously synthesizing oleanolic acid and protoalcohol (Dai et al., 2014). With the support of CRISPR technology, multiple sites can be modified concurrently, significantly reducing operational complexity and facilitating research related to the secondary metabolism of active substances.

The CRISPR system-mediated transcriptional regulation represents a promising strategy for manipulating plant metabolic pathways, serving as a complement to enzymatic regulation. This approach offers the potential to overcome challenges associated with multienzyme regulation in plant secondary metabolic pathways. By enabling CRISPR-mediated control of transcription factors, researchers gain a novel method for investigating the biosynthetic pathways of plant secondary metabolites. Transcriptional regulation primarily encompasses activation and repression mechanisms. In 2015, the C-terminus of the inactive Cas9 protein was fused with the VP64 transcriptional activation domain, resulting in the creation of a CRISPR-mediated transcriptional activator. This innovation has been successfully implemented in Arabidopsis, rice, and tobacco, demonstrating its functional efficacy (Lowder et al., 2015). Additionally, the 9-dCas PCO-3X (SRDX) synthetic transcriptional repressor has been utilized to repress both non-coding and protein-coding genes in Arabidopsis (Tang et al., 2017). Notably, this strategy enabled targeted mutagenesis of the SlAN2 gene, an R2R3-MYB transcription factor linked to anthocyanin biosynthesis in the tomato genome, thereby confirming the role of SlAN2 (Zhi et al., 2020). Transcription factors can manipulate metabolism by binding to cis-elements in promoter regions, thereby inhibiting or activating the expression of gene-encoded enzymes. For instance, in jasmonate biosynthesis, the transcription factor CrMYC2 directly binds to the cis-element in the promoter of the ORCA3 gene, regulating genes associated with terpene alkaloid biosynthesis. The CRISPR-mediated modification of the ORCA3 promoter cis-element has been proposed as a strategy to enhance terpene alkaloid biosynthesis (Zhang et al., 2011). Additionally, CRISPR-a can be utilized to simultaneously activate multiple transcripts of factors, further promoting the synthesis of secondary metabolites. This approach enables researchers to modulate complex metabolic pathways and increase the content of medicinal components in plants. Transcriptional regulation is influenced not only by transcription factors but also by signal transduction pathways. Plants typically respond to environmental stimuli through a series of signaling molecules. By employing CRISPR technology, researchers can interfere with these signaling pathways, thereby affecting the synthesis of secondary metabolites. For example, editing genes involved in a plant’s stress response can enhance the plant’s resilience while improving its ability to synthesize medicinal ingredients. Recent advancements in the transcriptional regulation of plant secondary metabolism have underscored the pivotal role of CRISPR gene editing technology in promoting research in this field and exploring pathways to human health.

Signaling pathways play a crucial role in plant growth, development, and secondary metabolism. By sensing environmental changes, plants regulate their internal signaling pathways, which in turn affects the synthesis of secondary metabolites. Under stress conditions such as drought, salinity, and alkalinity, plants activate specific signaling pathways to adapt to their environment. These pathways often involve the synthesis and signal transduction of hormones, including abscisic acid and cytokinins. For instance, CRISPR technology can be employed to edit genes associated with abscisic acid synthesis, thereby enhancing plant resistance to stress and promoting the synthesis of related secondary metabolites (Guo M. et al., 2022). The role of plant hormones in secondary metabolism is significant and should not be overlooked. Through CRISPR technology, scientists can disrupt hormone signal transduction pathways and regulate plant responses to hormones, thus influencing the accumulation of secondary metabolites. Manipulating the balance between auxin and cytokinin can affect plant growth patterns and the synthesis of active compounds (Al Aboud, 2024). The synthesis of secondary metabolites typically results from intricate interactions among multiple signaling pathways. The application of CRISPR technology enables researchers to unravel these complex interaction networks and identify key genes and signaling pathways. In certain medicinal plants, studies have demonstrated cross-regulation among different signaling pathways. By utilizing CRISPR technology, these critical nodes can be precisely edited to optimize the production of secondary metabolites (Guo M. et al., 2022; Kumar et al., 2023).

Salvianolic acid is a significant active component of Salvia miltiorrhiza, renowned for its medicinal properties, which includepromoting blood circulation, alleviating blood stasis, relieving pain, clearing heat, cooling the blood, and eliminating carbuncles. This compound holds considerable medicinal and economic value (Liang et al., 2016). The biosynthesis of phenolic acid in Salvia miltiorrhiza occurs via the phenylpropanoid pathway, with research primarily focusing on the functional enzymes involved in tanshinones and phenolic acid. The researchers employed CRISPR technology to target the RAS gene, which plays a crucial role in phenolic acid synthase activity, achieving a mutation rate of approximately 50%. This led to a corresponding decrease in both phenolic acid content and RAS expression levels (Zhou et al., 2018). Similar studies were conducted on the knockout of the SmLACs gene to investigate their role in phenolic acid metabolism, revealing that the growth of hairy roots was significantly inhibited and that lignin content was nearly undetectable, alongside a reduction in the accumulation of rosmarinic acid and salvianolic acid B (Zhou et al., 2021). Furthermore, the researchers utilized CRISPR technology to examine the SmPAL1 and SmLAC gene families in the secondary metabolism of Salvia miltiorrhiza (Feng, 2019, 58-70; Qiu, 2018, 32-38). These findings illustrate that the CRISPR/Cas9 system can effectively identify important genes within plant gene families and serves as an efficient and specific genome editing tool for Salvia miltiorrhiza. As a medicinal plant with a well-characterized genome, Salvia miltiorrhiza possesses a mature genetic transformation system, which offers a promising avenue for future research on metabolism of active substance using CRISPR technology. Recently, an enhanced gene transformation system known as Cut-Dip-Budding (CDB) has been developed, effectively promoting the application of CRISPR/Cas9 in gene functional analysis and breeding (Cao et al., 2024). Additionally, a protoplast-plant regeneration system for Salvia miltiorrhiza has been successfully established, enabling gene-free CRISPR/Cas9-mediated gene editing that can target one or multiple sites (Hsu et al., 2024). The advancement of plant genome editing technology now benefits from a streamlined and efficient genetic transformation systems, which promise to accelerate the discovery and enhancement of functional genes, particularly for medicinal plants that have traditionally posed challenges for transformation. CRISPR technology has the potential to significantly lower production costs per unit of product by increasing throughput and reducing production time. Conventional methods for extracting salvia acid often necessitate substantial quantities of raw materials and involve complex extraction processes. In contrast, genetic engineering allows for the synthesis of the same compounds in microorganisms, thereby decreasing reliance on natural plant sources. An optimized biosynthetic pathway can yield higher quality and greater quantities of salvianic acid, thereby meeting market demand. These advancements have notably enhanced the medicinal value of Salvia miltiorrhiza and its application in the treatment of cardiovascular diseases.

Anthocyanins are common secondary metabolites found in plants, known for their antioxidant and free radical scavenging properties. They are widely utilized in the food, pharmaceutical and beauty industries (Vimolmangkang et al., 2013). Plants serve as the primary source for anthocyanin extraction, and numerous studies have investigated the application of CRISPR technology to regulate the anthocyanin content in these organisms. For instance, CRISPR technology was employed to knock out the VvbZIP36 gene, identified as a negative regulator of anthocyanin accumulation within the grape bZIP family, thus promoting the accumulation of related metabolites (Tu et al., 2022). Additionally, CRISPR was utilized to induce targeted mutagenesis of the tomato gene SlAN2, an R2R3-MYB transcription factor associated with anthocyanin biosynthesis in the tomato genome. The study revealed that, compared to wild-type plants, the fruit weight and anthocyanin content of the mutant were reduced, thereby confirming the role of SlAN2 in regulating anthocyanin accumulation (Zhi et al., 2020). Beyond grapes and tomatoes, the CRISPR/Cas9 system successfully edited three key genes in the anthocyanin biosynthetic pathway of black rice—OsF3H, OsDFR and OsLDOX—with an editing efficiency of 56.7%. This targeted modification led to significant changes in seed color and anthocyanin content in the mutant strains (Jung et al., 2019). These results demonstrate that the CRISPR/Cas9 system can effectively induce substantial gene-specific mutations in black rice, suggesting its potential application in the breeding of crops and medicinal plants in the future. CRISPRi technology-mediated silencing of the transcriptional repressor MetJ can relieve the inhibition of the methionine biosynthetic pathway, thereby increasing the availability of S-adenosylmethionine (SAM) and promoting the production of O-methylated anthocyanins, with output increasing to 51 mg/L. This result represents a two-fold improvement compared to the non-targeting CRISPRi control strain, and an overall enhancement of 21-fold (Cress et al., 2017). To detect the presence of the transgene, the researchers combined CRISPR/Cas9 gene-editing technology with a system that activates anthocyanin biosynthesis, providing a visible signature. This anthocyanin tag-assisted CRISPR (AAC) technology enables the identification of transgenic events at the callus stage, the selection of transformants with high Cas9 expression, and the identification of non-transgenic plants in the field. The researchers utilized AAC technology to edit the LAZY1 and G1 genes, successfully generating numerous transgenic-free and targeted gene-edited plants in the T1 generation, thereby significantly reducing the labor, time, and cost associated with editing target genes in rice (He et al., 2020). Anthocyanins are directly associated with plant coloration, prompting some studies to utilize the CRISPR/Cas9 system to investigate the underlying mechanisms responsible for the blue flowers observed in Japanese gentian plants, specifically the accumulation of polyacylated anthocyanins known as gentiodelphin. By knocking out three anthocyanin-modifying genes (Gt5GT, Gt3′GT, and Gt5/3′AT), the flower colors produced by the transgenic lines exhibit variations of light reddish-purple, light pink, and lavender, respectively, which contrast with the bright blue of wild-type plants. The differences in flower coloration are significant. The glycosylation and subsequent acylation of the 3′-hydroxyl group on the B ring are essential for the development of blue gentian flowers (Tasaki et al., 2019). Furthermore, CRISPR/Cas9-mediated multiple gene editing technology can also be employed to target the PSY1, MYB12, and SGR1 genes associated with fruit color formation in tomatoes. This approach has successfully transformed the red-fruited tomato variety ‘Ailsa Craig’ into tomatoes exhibiting a range of fruit colors, including yellow, brown, pink, light yellow, pinkish brown, chartreuse, and light green. This strategy is more time-efficient than traditional breeding methods and yields GMO-free plants with varied fruit colors in less than a year (Yang et al., 2022a). Through precise gene editing, researchers can effectively enhance the anthocyanin content in plants and elevate fruit quality. As technology continues to advance, CRISPR technology is expected to play an increasingly significant role in future plant breeding, offering new solutions for the sustainable development of global agriculture.

Flavonoids represent a significant class of natural compounds that are abundantly present in various plants, particularly in fruits, vegetables, grains, bark, roots, stems, flowers, tea, and wine. These compounds exhibit diverse phenolic structures and frequently play crucial roles in plant growth, development, and resistance to environmental stressors. Furthermore, flavonoids are recognized for their potential health benefits in humans, exhibiting biological activities such as antioxidant, anti-inflammatory, and anti-cancer properties (Chen et al., 2023). Rutin, a natural flavonoid glycoside commonly referred to as vitamin P, possesses anti-allergic, antioxidant, anti-inflammatory, and antiviral properties (Yang et al., 2022b). As a valuable component of traditional Chinese herbal medicine, buckwheat is particularly rich in rutin and serves as its primary source. By employing CRISPR/Cas9-mediated targeted mutagenesis of the FtMYB45 gene, researchers achieved a mutation rate of 50%, resulting in increased levels of rutin, catechins, and other flavonoids in the trichome mutants (Wen et al., 2022). This innovative technology paves the way for advancements in functional gene research and the genetic enhancement of buckwheat. The GmF3H1, GmF3H2, and GmFNSII-1 genes in soybeans, which are involved in the flavonoid biosynthetic pathway, successfully increased the content of soybean isoflavones through CRISPR/Cas9-mediated multiple gene editing and enhanced resistance to soybean mosaic virus (SMV) (Zhang et al., 2020). Concurrently, the key flavonoid compound UDP-2-O-glucose transferase (MdPGT1) in apples can be knocked out using CRISPR/Cas9 gene editing technology, which reduces the accumulation of the flavonoid phloridzin in apple leaves without adversely affecting plant growth. This outcome sharply contrasts with the growth inhibition and leaf morphological changes induced by traditional transgenic methods, such as RNA interference (RNAi), when MdPGT1 is downregulated. Furthermore, the differential regulation of phytohormones may be associated with the effects of phloridzin reduction on growth (Miranda et al., 2023). The flavonoid biosynthetic pathway genes in rapeseed (Brassica napus L.) have also been studied using CRISPR/Cas9 technology to investigate their roles in seed color, oil content, and fatty acid composition. Mutations in these genes lead to alterations in seed color, an increase in seed oil content, and a reduction in the accumulation of pigments and lignin in the seed coat, which may facilitate further rapeseed breeding (Li et al., 2024). Additionally, CRISPR interference (CRISPRi) technology has been applied to downregulate the expression of the cinnamic acid-4-hydroxylase (C4H) gene in tobacco, where C4H serves as a key enzyme in the flavonoid biosynthetic pathway. Silencing the C4H gene using CRISPRi technology can enhance flavonoid biosynthesis in tobacco (Karlson et al., 2022). Additionally, multiple knockout mutant lines of the tomato HQT gene were generated through the CRISPR/Cas9 system. These slhqt (Solanum lycopersicum HQT knockout) mutants do not accumulate CGA or other caffeoylquinic acids (CQAs) in various parts of the plant, including fruits, stems, leaves, flowers, and roots. This finding suggests that CQA biosynthesis in tomato is predominantly reliant on the HQT pathway, a dependence that may also extend to other Solanaceae crops. Furthermore, the study revealed that the absence of CGA in slhqt mutant plants resulted in elevated levels of hydroxycinnamoyl-glucose and flavonoids compared to control plants (D'Orso et al., 2023). These results provide valuable insights into the role of HQT in plants and may have significant implications for crop improvement and stress response research.

Tanshinone is the primary lipophilic bioactive component of the Chinese herbal medicine derived from the rhizome of Salvia miltiorrhiza, serving as a significant intersection between traditional Chinese medicine and advanced molecular biology. Research has demonstrated that Tanshinone exhibits a range of biological activities, including antibiotic properties, as well as anti-inflammatory and antioxidant effects (Gao et al., 2009). Preparations of sage are currently undergoing phase II clinical trials for the treatment of cardiovascular disease (Chen et al., 2014). Since its initial application in 2017, CRISPR technology has been employed to knock out the SmCPS1 gene inSalvia miltiorrhiza, facilitating the investigation of the gene’s role in the tanshinone and the changes in tanshinone production pathway and alterations in tanshinone content. This represents the first instance of CRISPR technology being utilized to study Salvia miltiorrhiza, heralding a new era in the exploration of metabolic processes (Li et al., 2017). Additionally, this technology has been applied to knock out the enzyme-related genes SmCYP76AK2 and SmCYP76AK3 in Salvia miltiorrhiza, with the objective of examining their influence on tanshinone synthesis (Li et al., 2021). Subsequently, CRISPR technology successfully knocked out the SmCYP76AK5 gene, which is involved in the tanshinone biosynthesis pathway, revealing a reduction in tanshinone levels in mutant plants (Yu, 2019, 34-40). Studies have demonstrated that the SmMYB98 transcription factor is highly expressed in the lateral roots of Salvia miltiorrhiza and can be either knocked out (KO) or overexpressed (OE) using CRISPR/Cas9 technology. Experimental results indicate that the overexpression of SmMYB98 leads to an increase in the accumulation of Tanshinones and Salvianolic acids, whereas knockout of this factor results in a reduction of these metabolites. Furthermore, SmMYB98 also plays a negative regulatory role in gibberellin biosynthesis (Hao et al., 2020). Single gene editing in plants utilizing CRISPR technology is primarily focused on secondary metabolic systems. Notably, the same active substance can be associated with different metabolic pathways, which often involve complex regulatory relationships among enzymes. Moreover, a single enzyme may be regulated by multiple genes. CRISPR stands out as a highly efficient and rapid gene editing technology, demonstrating significant potential in the realm of multigene editing. The principle underlying CRISPR multiple gene editing involves constructing multiple sgRNAs within a single vector for transformation and expression, achieved through techniques such asGolden Gate cloning or Gibson assembly. These sgRNAs are then combined with the Cas9 protein to simultaneously target and knock down multiple genes using CRISPR technology (Ma et al., 2016). Researchers have reported employing the Golden Gate assembly strategy to construct sgRNAs targeting the Salvia genes SmDML3 and SmMET1 within the same vector. This approach utilizes CRISPR/Cas9 technology to achieve a double knockdown of these genes, demonstrating higher mutation efficiency compared to single-gene editing with CRISPR (Li, 2018, 122-129). The metabolome and transcriptome were analyzed by comparing two red root systems and one white root system of Salvia miltiorrhiza. The study found that the content of 18 Tanshinones in the white roots was significantly lower than that in the other roots, and the expression of 5 genes related to Tanshinone biosynthesis was significantly down-regulated in white roots, thereby affecting the metabolic flow of Tanshinone. The research speculates that this may explain why exhibits white roots. These findings not only elucidate the formation mechanism of root color in Salvia, but also provide an effective approach for exploring the biosynthetic pathway of Tanshinone. This study revealed the potential mechanisms underlying Tanshinone biosynthesis in Salvia miltiorrhiza by integrating metabolome and transcriptome analyses, and employed CRISPR/Cas9 technology to investigate and validate the functions of related genes, thereby establishing a foundation for genetic improvement and synthetic biology strategies in Salvia miltiorrhiza (Su et al., 2021). CRISPR technology holds significant potential for enhancing the secondary metabolism pathways of tanshinone. By employing gene editing, researchers can reconstruct metabolic pathways and explore regulatory mechanisms, thereby optimizing the synthesis of tanshinone and increasing both its yield and medicinal value. Furthermore, the ongoing advancement of this technology is expected to unlock even greater opportunities for the future of plant metabolic engineering, facilitating more efficient biosynthesis and resource utilization.

Malaria is a globally prevalent insect-borne disease, with the highest incidence occurring in Africa (Hu et al., 2021). Artemisinin, an active component derived from the plant Artemisia annua, is effective in treating malaria and has also been reported to possess antiviral, anticancer, and anti-schistosomiasis properties. This compound has been the subject of extensive research, including investigations utilizing CRISPR technology (Abdin et al., 2003). Specifically, CRISPR/Cas9 has been employed to enhance artemisinin content by disrupting the sterol synthesis pathway (Koerniati and Simanjuntak, 2020). Both artemisinin and sterols are synthesized from the common precursor farnesyl diphosphate (FDP). The gene squalene synthase (SQS), which regulates sterol biosynthesis, is considered a competing gene in artemisinin biosynthesis. The strategy of promoting artemisinin production by employing CRISPR to knock out the SQS gene. Currently, the primary sources for artemisinin extraction are the herb Artemisia annua and microbial cultures. The artemisinin biosynthetic pathway in Bacillus subtilis is compartmentalized into three distinct modules: a terpene synthase module, a branching pathway module, and a central metabolic module. The researchers utilized CRISPR technology to enhance genes related to artemisinin synthesis, targeting the TCA cycle, MEP pathway, branched pathway, and terpene synthase pathway. This strategy offers a novel approach to increasing terpenoid content in Bacillus subtilis (Song et al., 2021). Researchers are investigating transcription factors that play a crucial regulatory role in the biosynthesis of artemisinin and flavonoids. They employ CRISPR/Cas9 technology to edit the R2R3-MYB transcription factor encoded by the AaTAR2 gene. Knocking out AaTAR2 significantly reduces artemisinin content, while its overexpression leads to a notable increase in artemisinin levels. This indicates that AaTAR2 serves a positive regulatory function in artemisinin biosynthesis and may influence artemisinin production by affecting the development of glandular secretory hairs (Zhou et al., 2020). As noted by Ikram and Simonsen, scientists are exploring various bioengineering techniques to enhance the yield of Artemisia annua. These techniques encompass genetic modification of A. annua itself and heterologous expression in other plant systems, such as tobacco and mosses (Ikram and Simonsen, 2017). The primary focus of these efforts has been on enhancing the expression of key enzymes in the artemisinin biosynthetic pathway and inhibiting competing pathways through metabolic engineering, thereby redirecting more metabolic flow toward artemisinin synthesis. These advancements not only offer new strategies for artemisinin production but also provide valuable insights and methodologies for the production of other plant-derived natural products.

The triterpenoid ganoderic acids (GAs) derived from Ganoderma lucidum represent a valuable component of traditional Chinese herbal medicine, known for their anti-tumor properties. Additionally, GAs serve as critical indicators of the quality of Ganoderma lucidum and its associated products. Researchers have successfully developed a CRISPR/Cas9 editing system aimed at functional genes involved in GA biosynthesis, resulting in a significantly increase in the GA content (Wang et al., 2020). This technology is also employed to knock out key genes within the GA biosynthesis pathway, including CYP5150L8, as well as the homologous genes glcrz1 and glcrz2, which are associated with the calcium signal transduction factor CRZ1. The objective is to elucidate their roles in the influence of calcium signaling on GA biosynthesis. The study revealed that, in comparison to the wild-type strain, the glcrz1-knockout strain did not exhibit increased GA production following the addition of Ca²⁺. Conversely, the glcrz2-knockout strain demonstrated severely impaired hyphal growth and GA synthesis upon Ca²⁺ supplementation. (Zhang Y. et al., 2023; Li and Zhong, 2020) These findings hold significant implications for molecular breeding and biotechnological applications of Ganoderma lucidum, offering substantial potential to inform future research and developments in this area.

Triterpene saponins, a class of steroidal compounds recognized for their anticancer effects, are commonly referred to as ginsenosides. However, their natural abundance in ginseng is significantly limited. Consequently, enhancing the accumulation of triterpene saponins through gene editing techniques has become a major focus of research on secondary metabolites in ginseng (Sparg et al., 2004). In 2022, researchers reported the groundbreaking application of the CRISPR/Cas9 system to induce targeted mutations in the ginsenoside biosynthetic pathway (Choi et al., 2022). They constructed a PPT-type ginsenoside-deficient mutant, which produced only PPD-type saponin through CRISPR/cas9-mediated mutagenesis of the ginseng PPT synthase gene. PPT synthase in ginseng catalyzes the conversion of PPD saponins into PPT saponins, and significant differences exist in the pharmacological activities between these two types of saponins. Therefore, novel pure PPD ginseng mutants may offer new medicinal value compared to wild-type ginseng. In the same year, a CRISPR-mediated regulatory strategy for the ginsenosides biosynthesis pathway was also reported. This study combined the overexpression of ginsenoside synthesis genes squalene cyclooxygenase, Pq3-O-UGT2 and PAL with CRISPR/cas9 knockdown of CYP716A53v2 to block branched shunting in the ginsenoside production pathway, thereby enhancing the accumulation of ginsenoside Rg3 (Yao et al., 2022). Triterpene saponins are not only abundant in ginseng but also play a significant role in the active substances of alfalfa plants.CRISPR/cas9 technology was employed to knock out CYP93E2 and CYP72A61 genes associated with soybean saponinol B biosynthesis in alfalfa plants, achieving an editing efficiency of 84% (Confalonieri et al., 2021). The results indicate that all CYP93E2 knockout mutants do not produce saponin elements, instead shifting metabolic flux to generate valuable saponin compounds. Furthermore, studies utilizing CRISPR-Cas9 technology have targeted the glycosyltransferase gene (GuCSyGT) derived from cellulose synthase in licorice. The complete deletion of soybean saponin I confirms the role of GuCSyGT in the biosynthesis of triterpene saponins in plants, providing new insights for studying the physiological functions of plant saponins (Sakanishi et al., 2023).

Paclitaxel, a complex diterpene alkaloid with inherent anticancer properties, has emerged as a prominent active compound in medical research in recent years (Weaver, 2014). CRISPR technology has significantly contributed to the investigation of paclitaxel biosynthesis and drug resistance. Specifically, researchers utilized CRISPR-guided DNA methylation technology to knock down phenylalanine ammonia lyase (PAL), which is the initial enzyme in the phenylpropanoid pathway that regulates paclitaxel biosynthesis in yew, resulting in a notable increase in paclitaxel accumulation (Brzycki Newton et al., 2023). Additionally, in triple-negative breast cancer (TNBC) cell models, scientists identified multiple gene targets associated with paclitaxel resistance, including ATP8B3, FOXR2, FRG2, and HIST1H4A, through a combination of in vitro and in vivo genome-wide CRISPR screening methods. The deletion of these genes was found to enhance TNBC cell resistance to paclitaxel and has been recognized as a negative regulator of cancer stem cell properties (Yan et al., 2023). Furthermore, researchers employed the CRISPR/Cas9 system to integrate various GGPP synthase genes into the yeast genome, establishing a biotechnological platform for the synthesis of paclitaxel biosynthetic precursors. This engineered strain may facilitate the identification of candidate genes for future microbial paclitaxel biosynthesis (Utomo et al., 2021). Collectively, these studies underscore the substantial potential of CRISPR technology to augment paclitaxel production and address challenges related to cancer drug resistance.

Cocaine and morphine are primarily derived from the opium poppy, which is recognized in traditional Chinese medicine for its properties that strengthen the spleen, enhance appetite, clear heat, and detoxify. It is commonly employed in the treatment of diarrhea, dysentery, and reflux (Kang and Wang, 2011). In 2016, CRISPR technology was first applied to the opium poppy, utilizing viral TRV to successfully knock out the 4′OMT2 gene and deliver the CRISPR/Cas9 complex into target cells. This innovative method was employed to investigate the function of the 4′OMT2 gene within the biosynthetic pathway of pharmaceutically active substances in the opium poppy, leading to a significant reduction in the production of cocaine and morphine in the resulting mutant plants (Alagoz et al., 2016). Nicotine is a compound characterized by a unique structure and a range of biological activities. It exerts diverse effects on the nervous system, influencing addiction, anxiety, cognitive function, and various other aspects. While it holds potential for certain medical applications, nicotine is also associated with significant health risks. Research on nicotine not only enhances our understanding of its biological effects but also yields crucial insights for public health policy and smoking cessation treatments (Sansone et al., 2023). Specifically, CRISPR/Cas9-induced mutations in the A622 and BBL genes have a marked effect on nicotine biosynthesis in tobacco. These mutations not only reduce the levels of nicotine and other alkaloids but also result in developmental abnormalities in the plants (Jeong et al., 2024). Additionally, the establishment and application of the CRISPR/Cas9 system in wild tobacco (Nicotiana alata) have led to significant advancements in genetic transformation methods. By optimizing these methods, we achieved a notable increase in genetic transformation efficiency and successfully performed genome editing in N. alata (Yuan et al., 2024). Furthermore, an optimized CRISPR/Cas9 system was employed to enhance gene editing efficiency in tobacco. By utilizing the OsU3-tRNA promoter combination to drive sgRNA expression and incorporating a visible Ros1 expression cassette for monitoring transgenic events, we simplified the screening process and improved overall efficiency (Zhang J. et al., 2023).

Glycosides are a class of compounds commonly found in plants, comprising sugar molecules and non-sugar moieties, referred to as aglycones, which are linked by glycosidic bonds. These compounds can be categorized into various groups based on the type of sugar and the characteristics of the aglycone. Due to their diverse biological activities, glycosides play a significant role in drug development and therapeutic applications (Kytidou et al., 2020). Transgenic technology and CRISPR-Cas9 gene editing were employed to knock out the MYB28 gene in broccoli, resulting in a reduction of glucoside accumulation (Zhang et al., 2024). This demonstrates that CRISPR-Cas9 technology can effectively induce mutations in specific genes within the broccoli genome, which is significant for further validating gene functions and achieving precise trait improvement. Additionally, the BoaAOP2 gene in Chinese kale was edited using the CRISPR/Cas9 system. This gene encodes a 2-oxoglutarate-dependent dioxygenase that catalyzes the conversion of glucoside (GRA) into gluconaphthyridine (GNA). In the edited T1 generation mutant, the GRA content increased significantly, while the GNA content and total aliphatic glucosinolate (GSL) levels decreased. These findings suggest that targeted editing of the BoaAOP2 gene via CRISPR/Cas9 alters the metabolic flux of aliphatic GSL side chains and enhances GRA content in Chinese kale (Zheng et al., 2023). A high-efficiency broccoli genetic transformation system mediated by PEG-Ca²⁺ was utilized to investigate the subcellular localization of the FMOGS-OX5 gene, which is associated with glucoside synthesis, as well as the clubroot resistance gene CRa. Both genes are expressed in the cytoplasm and nucleus, providing a scientific basis for studying the regulation of glucoside metabolism and clubroot disease resistance in cruciferous crops (Zhao et al., 2023). Utilizing the CRISPR/Cas9 system, the phytoene desaturase (BoPDS) gene in cabbage, which is critical to the carotenoid biosynthetic pathway, was edited. The observation of albino seedlings in the T0 generation suggests that the BoPDS gene may have been successfully edited. Additionally, mutations in the Bol016089 gene, which shares high homology with the BoPDS gene, were detected, indicating that the CRISPR/Cas9 system can edit multiple genes simultaneously (Ma et al., 2019). By employing the CRISPR/Cas9 system alongside the endogenous tRNA processing system, multiple genes in cabbage—including BoPDS, BoSRK, and BoMS1—were edited to achieve multi-site and multi-gene mutations. Notably, mutations in the BoSRK3 gene completely suppressed self-incompatibility, transforming self-incompatible lines into self-compatible lines. Furthermore, mutations in the BoMS1 gene resulted in completely male-sterile mutants, which can be economically propagated through bee-mediated cross-pollination (Ma et al., 2019).

Polyketides represent a substantial class of structurally diverse secondary metabolites that are prevalent in microorganisms, plants, and animals. These compounds play significant ecological roles within organisms and are extensively utilized in medicine, agriculture, industry, and other domains owing to their varied biological activities. The CRISPR interference (CRISPRi) system is employed to finely regulate central metabolic pathways in Escherichia coli, thereby enhancing polyketide biosynthesis. By silencing candidate genes using synthetic sgRNA, intracellular levels of malonyl-CoA were successfully increased, leading to an augmentation in the production of the plant-specific secondary metabolite (2S)-naringenin (Wu et al., 2015). The CRISPR-Cas9 system facilitates metabolic engineering by enabling the deletion and insertion of biosynthetic gene clusters (BGCs) to enhance polyketide production. Additionally, the CRISPR-BEST (CRISPR-Base Editing System) has been developed for efficient base editing aimed at promoting polyketide synthesis (Yang et al., 2021). The CRISPR-Cas9 system was utilized to knock out the erythromycin biosynthetic gene cluster, resulting in the generation of a recombinant strain, Abery, which inhibits the synthesis of erythromycin while accumulating significant amounts of the synthetic substrates methylmalonate-CoA and malonate coenzyme A. Using Abery as a heterologous host, three genes—AsCHS, RgTAL, and Sc4CL—were introduced to facilitate the production of new polyketides from L-tyrosine and methylmalonate-CoA (Ren et al., 2021). And a review discusses the application of CRISPR/Cas9 technology in the synthesis of secondary metabolites, including polyketides, in filamentous fungi. Furthermore, CRISPR/Cas9 technology has been employed to enhance the production of polyketides, such as increasing the production of the plant growth hormone gibberellic acids through gene editing. The researchers also explored the potential of utilizing CRISPR/Cas9 technology to activate silent gene clusters within the polyketide biosynthetic pathway (Jiang et al., 2021). A customized pCRISPR–Cas9apre system was developed to enhance the biosynthesis of ansamitocin P-3 (AP-3), a polyketide, in Actinosynnema pretiosum. Through CRISPR–Cas9-mediated gene editing, the researchers deleted gene clusters that compete with the AP-3 biosynthetic pathway and enhanced the supply of “glycolate” elongation units by inserting bidirectional promoters (BDPs). These strategies successfully increased AP-3 production (Guo S. et al., 2022). Additionally, the researchers established an efficient CRISPR/Cas9-mediated gene knockout system for Trichoderma hamatum T21, a filamentous fungus. They effectively knocked out the polyketide synthase gene (PKS) associated with pigment synthesis, facilitating the investigation of the fungus’s biological control mechanisms and the synthesis of secondary metabolites. The article discusses methods for editing the type I polyketide synthase (PKS) gene in vitro using the CRISPR-Cas9 system. The researchers successfully heterologously expressed the edited biosynthetic gene cluster in E. coli, resulting in the production of the desired polyketide derivatives. This technology provides a platform for the rational design of natural product derivatives for future drug development (Luo et al., 2023; Kudo et al., 2020). Furthermore, an article reviews the application of the CRISPR/Cas system in Streptomyces, with a particular emphasis on the identification and activation of silent polyketide biosynthetic gene clusters (smBGCs) through CRISPR/Cas technology. It summarizes the application of the CRISPR/Cas system in Streptomyces and discusses its potential for natural product discovery and production (Lee et al., 2024).

The application of CRISPR technology in plant secondary metabolites offers innovative solutions for the sustainable development of agriculture. By enhancing crop disease resistance, optimizing nutritional composition, and improving stress tolerance, CRISPR technology not only increases agricultural production efficiency but also contributes significantly to environmental protection and ecological balance. Looking ahead, as technology continues to advance, CRISPR is poised to play an increasingly vital role in the sustainable development of global agriculture.

The secondary metabolism network of active substances in medicinal plants often necessitates interactions between different pathways, leading to the production of medicinal active substances being influenced by the expression levels of multiple genes within these pathways. To further analyze the impact of gene networks on secondary metabolites or to enhance yields more precisely, it is essential to fine-tune the activities of relevant intermediate enzymes, ensuring that each reaction is compatible with the overall biosynthetic pathway. Currently, CRISPR-based strategies for secondary metabolite research typically involve knocking out or significantly altering the expression of target genes, rather than enabling precise regulation. However, researchers are now capable of fine-tuning gene activity in model plants. For instance, by inducing various mutations in non-coding regions, such as uORF regions, 5’UTR, and 3’UTR, a range of product levels can be generated, resulting in plants with the desired product content (Huang et al., 2020; Wang et al., 2024; Xing et al., 2020; Xue et al., 2023; Zeng et al., 2020). Similarly, introducing a series of mutations in amino acids can lead to the fine-tuning of enzyme activity (Xu et al., 2021). These methods have been validated and applied in model plants and may also serve as important strategies for enhancing medicinal plants.

In comparison to non-coding sequence editing, CRISPRa/i and epigenetic editing facilitated by the CRISPR system offer a straightforward, direct, and flexible approach, particularly for the simultaneous regulation of multiple genes. Enhancing the precision of CRISPRa/i regulation levels and CRISPR-based epigenetic regulation—such as the modification of regulatory elements or the engineering of guide RNA structures—can facilitate the quantitative expression of target genes to manageable levels. Moreover, current strategies for regulating secondary metabolic networks typically involve the editing or modulation of a single or a limited number of intermediate enzymes, rather than a comprehensive series of genes within the entire network. With a deeper understanding of each biological process and the rapid advancements in multiplex gene editing technology, varying degrees of simultaneous gene regulation will address challenges in plant pharmaceutical engineering, enabling production levels that surpass those achievable through traditional methods.

Currently, our understanding of the gene networks responsible for the production of most active ingredients in botanicals remains limited, as the functions of many genes are still unknown. CRISPR-based high-throughput screening is poised to significantly enhance the discovery of gene functions through reverse genetic screening. The establishment of CRISPR knockout (CRISPR-KO), CRISPR activation (CRISPRa), and CRISPR interference (CRISPRi) libraries enables high-throughput knockout, activation, and knockdown of genes, thereby influencing product yields. By leveraging the consistent one-to-one correspondence between product yields and the sgRNA used in CRISPR, critical unknown genes can be rapidly identified. However, the successful application of CRISPR-based high-throughput screening is contingent upon the development of efficient genetic transformation systems. In the future, advancements in genetic transformation technology and plant cell culture methods are expected to enhance CRISPR-based high-throughput screening strategies, providing significant support for individual-level gene discovery, analysis, regulation, and the utilization of plant cell factories. This progress will greatly facilitate the optimization and modification of secondary metabolic pathways, as well as improve the capability to transfer gene pathways across different species (Liu et al., 2023; Shalem et al., 2015).

Currently, CRISPR technology has enabled the editing of large DNA fragments and chromosomes, offering a novel strategy for investigating the functions of genes associated with secondary metabolites. For instance, CRISPR-based tools for large deletions and replacements facilitate the deletion of gene clusters, as well as the exchange of promoters and genes. Furthermore, CRISPR can induce chromosomal rearrangements, including interchromosomal rearrangements, inversions, deletions, and truncations, allowing for the rapid simplification and modification of plant biological chassis. This capability can be employed to eliminate toxic by-products or redundant sequences, remove negative regulators that hinder cell or plant growth, or upregulate endogenous plant processes that enhance the production of secondary metabolites. Additionally, it accelerates the construction of minimal genomes or modifies them to be more suitable as biofactories. Building on the optimization of the chassis, CRISPR-based targeted large DNA sequence integration tools, such as the newly developed Prime Editing technology, can be utilized for the swift transfer of exogenous secondary metabolism-related elements or entire pathways to address deficiencies or key components in the chassis gene. This advancement facilitates the stable and efficient production of target metabolites in plant molecular agriculture (Sun et al., 2024; Zhu et al., 2022). Moreover, CRISPR-based DNA fragment manipulation technology allows for the straightforward testing and optimization of pathways, further enhancing the yield of molecular agriculture products. Consequently, the development of robust CRISPR-based systems for operating on large DNA fragments and their applications will be a significant aspect of CRISPR’s role in the biosynthesis of bioactive compounds from plants in the future.