Application of genome editing techniques to regulate gene expression in crops.

BACKGROUND: Enhanced agricultural production is urgently required to meet the food demands of the increasing global population. Abundant genetic diversity is expected to accelerate crop development. In particular, the development of the CRISPR/Cas genome editing technology has greatly enhanced our ability to improve crop's genetic diversity through direct artificial gene modification. However, recent studies have shown that most crop improvement efforts using CRISPR/Cas techniques have mainly focused on the coding regions, and there is a relatively lack of studies on the regulatory regions of gene expression. RESULTS: This review briefly summarizes the development of CRISPR/Cas system in the beginning. Subsequently, the importance of gene regulatory regions in plants is discussed. The review focuses on recent developments and applications of mutations in regulatory regions via CRISPR/Cas techniques in crop breeding. CONCLUSION: Finally, an outline of perspectives for future crop breeding using genome editing technologies is provided. This review provides new research insights for crop improvement using genome editing techniques.

Potentialities of Dandelion (Taraxacum Mongolicum Hand.-Mazz.) Flower Extracts on Gastric Protection against Helicobacter Pylori and Characterization of its Bioactive Constituents.

OBJECTIVES: Dandelion has been shown to exert anti-inflammatory and anti-bacterial effects. Our study aimed to identify the effect and mechanism of dandelion flower extracts on H. pylori-induced gastritis and screen for novel antimicrobial substances. METHODS: Anti-H. pylori activities of water extracts(WEDF) and ethanol extracts (EEDF) of dandelion flowers were performed with disk diffusion method assay, MIC, and MBC. The H. pylori-induced model was constructed to examine the gastroprotective of EEDF using RUT, pathological analysis, and ELISA. RESULTS: EEDF exhibited better anti- H. pylori and urease inhibition activities than WEDF. In vivo studies, EEDF can reduce the adhesion of H. pylori to the gastric mucosa, alleviate gastric damage, and concurrently reduce the levels of TNF-α and IL-6 in gastric tissues. The six phenolic compounds showed urease inhibition effect (IC50: 2.99±0.15 to 66.08±6.46 mmol/mL). Among them, chlorogenic acid, caffeic acid, and luteolin also had anti-H. pylori activity (MIC: 64-256 µg/mL). CONCLUSION: EEDF exhibited anti-H. pylori, gastroprotective and anti-inflammatory effects. Chicoric acid and luteolin may be the main active compounds of dandelion flowers to exert anti-H. pylori, and worthy of further investigation.

Stepwise Diagnostic Product Ions Filtering Strategy for Rapid Discovery of Diterpenoids in Scutellaria barbata Based on UHPLC-Q-Exactive-Orbitrap-MS.

Diterpenoids are considered the major bioactive components in Scutellaria barbata to treat cancer and inflammation, but few comprehensive profiling studies of diterpenoids have been reported. Herein, a stepwise diagnostic product ions (DPIs) filtering strategy for efficient and targeted profiling of diterpenoids in Scutellaria barbata was developed using UHPLC-Q-Exactive-Orbitrap-MS. After UHPLC-HRMS/MS analysis of six diterpenoid reference standards, fragmentation behaviors of these references were studied to provide DPIs. Then, stepwise DPIs filtering aimed to reduce the potential interferences of matrix ions and achieve more chromatographic peaks was conducted to rapidly screen the diterpenoids. The results demonstrated that stepwise DPIs were capable of simplifying the workload in data post-processing and the effective acquisition of low abundance compounds. Subsequently, DPIs and MS/MS fragment patterns were adopted to identify the targeted diterpenoids. As a result, 381 diterpenoids were unambiguously or tentatively identified, while 141 of them with completely new molecular weights were potential new diterpenoids for Scutellaria barbata. These results demonstrate that the developed stepwise DPIs filtering method could be employed as an efficient, reliable, and valuable strategy to screen and identify the diterpenoid profile in Scutellaria barbata. This might accelerate and simplify target constituent profiling from traditional Chinese medicine (TCM) extracts.

Gastroprotective action of the extract of Corydalis yanhusuo in Helicobacter pylori infection and its bioactive component, dehydrocorydaline.

ETHNOPHARMACOLOGICAL RELEVANCE: Helicobacter pylori (H. pylori) infection is a frequent chronic infection. Persistent infection is the strongest risk factor for developing gastric complications leading to gastric cancer. The antibiotic resistance of current anti-H. pylori drugs lead to the search for novel candidates from medicinal plants. Traditionally, Corydalis yanhusuo (Y.H. Chou & Chun C.Hsu) W.T. Wang ex Z.Y. Su & C.Y. Wu (Papaveraceae) has been used for the treatment of digestive system diseases in China. So, it's essential to explore and confirm the anti-H. pylori activity of C. yanhusuo and characterize the pharmacologically active compounds. AIM OF THE STUDY: This study aims to evaluate the efficacy of C. yanhusuo as complementary or alternative modes of treatment against H. pylori-related diseases and ascertain the active substances of C. yanhusuo to develop non-toxic, natural, and inexpensive products. MATERIALS AND METHODS: C. yanhusuo was subjected to solid-liquid extraction with water (WECY), ethanol EECY), and chloroform (CECY). The extracts were screened by agar diffusion assay, the minimum inhibitory concentrations (MIC), the minimum bactericidal (MBC) for their in vitro antimicrobial activity, and by Berthelot reaction for urease inhibition. To assess the in vivo action, H. pylori-induced C57BL/6 mice were used to detect RUT biopsy, perform visual and histopathological analyses and evaluate IgG expression. Furthermore, we compared the anti-H. pylori activities of major alkaloids in CECY to identify the bioactive constituents. RESULTS: Among the three C. yanhusuo extracts, CECY showed the maximum in vitro antibacterial activity. Administration of CECY significantly inhibited the survival of H. pylori colonized in the gastric mucosa and alleviated gastric damage along with a reduction in the expression levels of IgG in H. pylori-infected mice. Berberine and dehydrocorydaline exhibited obvious anti-H. pylori activity with MIC of 25 and 12.5 µg/mL, respectively. CONCLUSION: C. yanhusuo extracts showed anti-H. pylori activity in different degrees. Among them, CECY showed significant anti-H. pylori, gastroprotective and anti-inflammatory activities in vivo and in vitro. Dehydrocorydalmine, an active alkaloid compound isolated from C. yanhusuo, warranted further investigation for its potential anti-H. pylori activity.

The Development of Herbicide Resistance Crop Plants Using CRISPR/Cas9-Mediated Gene Editing.

The rapid increase in herbicide-resistant weeds creates a huge challenge to global food security because it can reduce crop production, causing considerable losses. Combined with a lack of novel herbicides, cultivating herbicide-resistant crops becomes an effective strategy to control weeds because of reduced crop phytotoxicity, and it expands the herbicidal spectrum. Recently developed clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas)-mediated genome editing techniques enable efficiently targeted modification and hold great potential in creating desired plants with herbicide resistance. In the present review, we briefly summarize the mechanism responsible for herbicide resistance in plants and then discuss the applications of traditional mutagenesis and transgenic breeding in cultivating herbicide-resistant crops. We mainly emphasize the development and use of CRISPR/Cas technology in herbicide-resistant crop improvement. Finally, we discuss the future applications of the CRISPR/Cas system for developing herbicide-resistant crops.

Generation of imidazolinone herbicide resistant trait in Arabidopsis.

Recently-emerged base editing technologies could create single base mutations at precise genomic positions without generation DNA double strand breaks. Herbicide resistant mutations have been successfully introduced to different plant species, including Arabidopsis, watermelon, wheat, potato and tomato via C to T (or G to A on the complementary strand) base editors (CBE) at the P197 position of endogenous acetolactate synthase (ALS) genes. Additionally, G to A conversion to another conserved amino acid S653 on ALS gene could confer tolerance to imidazolinone herbicides. However, no such mutation was successfully generated via CBE, likely due to the target C base is outside of the classic base editing window. Since CBE driven by egg cell (EC) specific promoter would re-edit the wild type alleles in egg cells and early embryos, we hypothesized the diversity of base editing outcomes could be largely increased at later generations to allow selection of desired herbicide resistant mutants. To test this hypothesis, we aimed to introduce C to T conversion to the complement strand of S653 codon at ALS gene, hosting a C at the 10th position within the 20-nt spacer sequence outside of the classic base editing window. While we did not detect base-edited T1 plants, efficient and diverse base edits emerged at later generations. Herbicide resistant mutants with different editing outcomes were recovered when T3 and T4 seeds were subject to herbicide selection. As expected, most herbicide resistant plants contained S653N mutation as a result of G10 to A10. Our results showed that CBE could create imidazolinone herbicide resistant trait in Arabidopsis and be potentially applied to crops to facilitate weed control.