Expanding plant genome editing scope and profiles with CRISPR-FrCas9 systems targeting palindromic TA sites.

CRISPR-Cas9 is widely used for genome editing, but its PAM sequence requirements limit its efficiency. In this study, we explore Faecalibaculum rodentium Cas9 (FrCas9) for plant genome editing, especially in rice. FrCas9 recognizes a concise 5'-NNTA-3' PAM, targeting more abundant palindromic TA sites in plant genomes than the 5'-NGG-3' PAM sites of the most popular SpCas9. FrCas9 shows cleavage activities at all tested 5'-NNTA-3' PAM sites with editing outcomes sharing the same characteristics of a typical CRISPR-Cas9 system. FrCas9 induces high-efficiency targeted mutagenesis in stable rice lines, readily generating biallelic mutants with expected phenotypes. We augment FrCas9's ability to generate larger deletions through fusion with the exonuclease, TREX2. TREX2-FrCas9 generates much larger deletions than FrCas9 without compromise in editing efficiency. We demonstrate TREX2-FrCas9 as an efficient tool for genetic knockout of a microRNA gene. Furthermore, FrCas9-derived cytosine base editors (CBEs) and adenine base editors (ABE) are developed to produce targeted C-to-T and A-to-G base edits in rice plants. Whole-genome sequencing-based off-target analysis suggests that FrCas9 is a highly specific nuclease. Expression of TREX2-FrCas9 in plants, however, causes detectable guide RNA-independent off-target mutations, mostly as single nucleotide variants (SNVs). Together, we have established an efficient CRISPR-FrCas9 system for targeted mutagenesis, large deletions, C-to-T base editing, and A-to-G base editing in plants. The simple palindromic TA motif in the PAM makes the CRISPR-FrCas9 system a promising tool for genome editing in plants with an expanded targeting scope.

Examining the impact of Tetragenococcus halophilus, Zygosaccharomyces rouxii, and Starmerella etchellsii on the quality of soy sauce: a comprehensive review of microbial population dynamics in fermentation.

Soy sauce is a popular fermented seasoning due to its distinct flavor and rich umami taste. Its traditional production involves two stages: solid-state fermentation and moromi (brine fermentation). During moromi, the dominant microbial population in the soy sauce mash changes, which is called microbial succession and is essential for the formation of soy sauce flavor compounds. Research has identified the sequence of succession, starting with Tetragenococcus halophilus, then Zygosaccharomyces rouxii, and lastly, Starmerella etchellsii. Factors such as the environment, microbial diversity, and interspecies relationships drive this process. Salt and ethanol tolerance influence microbial survival, while nutrients in the soy sauce mash support the cells in resisting external stress. Different microbial strains have varying abilities to survive and respond to external factors during fermentation, which impacts soy sauce quality. In this review, we would examine the factors behind the succession of common microbial populations in the soy sauce mash and explore how microbial succession affects soy sauce quality. The insights gained can help better manage the dynamic changes in microbes during fermentation, leading to improved production efficiency.

Epoxide Electroreduction.

Selective hydrogenation of epoxides would be a direct and powerful approach for alcohol synthesis, but it has proven to be elusive. Here, electrochemically epoxide hydrogenation using electrons and protons as reductants is reported. A wide range of primary, secondary, and tertiary alcohols can be achieved through selective Markovnikov or anti-Markovnikov ring opening in the absence of transition metals. Mechanistic investigations revealed that the regioselectivity is controlled by the thermodynamic stabilities of the in situ generated benzyl radicals for aryl-substituted epoxides and the kinetic tendency for Markovnikov selective ring opening for alkyl-substituted epoxides.

Loss of RBPMS in ovarian cancer compromises the efficacy of EGFR inhibitor gefitinib through activating HER2/AKT/mTOR/P70S6K signaling.

RBPMS may be a tumor suppressor in cancer, but its impact in modulation of drug sensitivity is unclear. This study aimed to investigate the regulatory role of RBPMS in cellular response to EGFR inhibitor gefitinib in ovarian cancer (OC). By western blotting assay, we revealed RBPMS was down-regulated in epithelial ovarian cancer tissues compared to normal control ovarian epithelial tissues. Overexpression of RBPMS inhibited cell viability and proliferation, and conferred gefitinib sensitivity, accompanied by reduced expression of p-EGFR, and vice versa. Proteomic analysis and flow cytometry experiments showed that RBPMS induced S-stage cell cycle arrest in gefitinib-treated OC cells. Co-IP assay suggested that HER2 was a downstream target of RBPMS, and RBPMS negatively regulated HER2 expression. HER2 counteracted the stimulation of RBPMS to cell growth blocking, gefitinib sensitivity and cell cycle arrest. We further demonstrated that RBPMS overexpression suppressed the activation of p-AKT, p-mTOR and p-P70S6K, which was rescued by up-regulation of HER2. The combination of AKT inhibitor MK2206 and gefitinib had a synergistic effect on OC cells with high level of RBPMS. In conclusion, through the direct inhibition of HER2/AKT/mTOR/P70S6K pathway, RBPMS may be a potential therapeutic target for improving gefitinib sensitivity in OC.

CRISPR-Cas9 mediated OsMIR168a knockout reveals its pleiotropy in rice.

MicroRNA168 (MIR168) is a key miRNA that targets the main RNA-induced silencing complex component Argonaute 1 (AGO1) to regulate plant growth and environmental stress responses. However, the regulatory functions of MIR168 need to be further elucidated in rice. In this paper, we generated clean OsMIR168a deletion mutants by CRISPR-Cas9 strategy. We then phenotypically and molecularly characterized these mutants. The rice OsMIR168a mutants grew rapidly at the seedling stage, produced more tillers and matured early. Compared to the wild-type plants, the mutants were shorter at maturity and produced smaller spikelets and seeds. Analysis of gene expression showed that the transcription levels of OsMIR168a's target genes such as OsAGO1a, OsAGO1b and OsAGO1d were elevated significantly in the OsMIR168a mutants. Intriguingly, OsAGO18, a member of a new AGO clade that is conserved in monocots, was confirmed to be a target of OsMIR168a not only by informatic prediction but also by expression analysis and a cell-based cleavage assay in the OsMIR168a mutants. Many protein-coding genes and miRNAs showed differential expression in the OsMIR168a mutants, suggesting OsMIR168a exerts a major transcriptional regulatory role, likely through its potential target genes such as OsAGO1s and OsAGO18. KEGG enrichment analysis of these differentially expressed genes pointed to OsMIR168a's involvement in important processes such as plant hormone signalling transduction and plant-pathogen interaction. These data collectively support that the complex regulation module of OsMIR168a-OsAGO1/OsAGO18-miRNAs-target genes contributes to agronomically important traits, which sheds light on miRNA-mediated crop breeding.

Genome-wide analyses of PAM-relaxed Cas9 genome editors reveal substantial off-target effects by ABE8e in rice.

PAM-relaxed Cas9 nucleases, cytosine base editors and adenine base editors are promising tools for precise genome editing in plants. However, their genome-wide off-target effects are largely unexplored. Here, we conduct whole-genome sequencing (WGS) analyses of transgenic plants edited by xCas9, Cas9-NGv1, Cas9-NG, SpRY, nCas9-NG-PmCDA1, nSpRY-PmCDA1 and nSpRY-ABE8e in rice. Our results reveal that Cas9 nuclease and base editors, when coupled with the same guide RNA (gRNA), prefer distinct gRNA-dependent off-target sites. De novo generated gRNAs by SpRY editors lead to additional, but insubstantial, off-target mutations. Strikingly, ABE8e results in ~500 genome-wide A-to-G off-target mutations at TA motif sites per transgenic plant. ABE8e's preference for the TA motif is also observed at the target sites. Finally, we investigate the timeline and mechanism of somaclonal variation due to tissue culture, which chiefly contributes to the background mutations. This study provides a comprehensive understanding on the scale and mechanisms of off-target and background mutations occurring during PAM-relaxed genome editing in plants.

CRISPR-BETS: a base-editing design tool for generating stop codons.

Cytosine base editors (CBEs) can install a predefined stop codon at the target site, representing a more predictable and neater method for creating genetic knockouts without altering the genome size. Due to the enhanced predictability of the editing outcomes, it is also more efficient to obtain homozygous mutants in the first generation. With the recent advancement of CBEs on improved editing activity, purify and specificity in plants and animals, base editing has become a more appealing technology for generating knockouts. However, there is a lack of design tools that can aid the adoption of CBEs for achieving such a purpose, especially in plants. Here, we developed a user-friendly design tool named CRISPR-BETS (base editing to stop), which helps with guide RNA (gRNA) design for introducing stop codons in the protein-coding genes of interest. We demonstrated in rice and tomato that CRISPR-BETS is easy-to-use, and its generated gRNAs are highly specific and efficient for generating stop codons and obtaining homozygous knockout lines. While we tailored the tool for the plant research community, CRISPR-BETS can also serve non-plant species.

Spin engineering of triangulenes and application for nano nonlinear optical materials design.

The recently synthesized triangulenes with non-bonding edge states could have broad potential applications in magnetics, spintronics and electro-optics if they have appropriate electronic structure modulation. In the present work, strategies based on molecular orbital theory through heteroatom doping are proposed to redistribute, reduce or eliminate the spin of triangulenes for novel functional materials design, and the role of B, N, NBN, and BNB in such intended electronic structure manipulation is scrutinized. π-Extended triangulenes with tunable electronic properties could be potential nonlinear optical (NLO) materials with appropriate inhibition of their polyradical nature. The elimination of spin is achieved by B, N, NBN, and BNB doping with the intended geometric arrangement for enhanced polarity. Intended doping of BNB results in an optimal structure with large static first hyperpolarizability (〈ß0〉) as well as strong Hyper-Rayleigh scattering (HRS) ßHRS(-2ω; ω, ω) (ω = 1064.0 nm), TG7-BNB-ba with a large 〈ß0〉 (18.85 × 10-30 esu per heavy atom) and ßHRS (1.15 × 10-28 esu per heavy atom) much larger than that of a synthesized triangular molecule (1.12 × 10-30 esu of 〈ß0〉 per heavy atom and 5.04 × 10-30 esu of ßHRS per heavy atom). The strong second order NLO responses in the near-infrared and visible regions, particularly the strong sum frequency generation, make these B or (and) N doped triangulenes promising candidates for the fabrication of novel carbon-based optoelectronic devices and micro-NLO devices.

Tuning the edge states in X-type carbon based molecules for applications in nonlinear optics.

Novel carbon based "X-type" graphene nanoribbons (GNRs) with azulenes were designed for applications in nonlinear optics in the present work, and the second order nonlinear optical (NLO) properties of those X-type GNRs were predicted using the sum-over-states (SOS) model. The GNRs with edge states are feasibly polarized. The effects of zigzag edges on the NLO properties of GNRs are scrutinized by passivation, and the electronic structures of GNRs are modulated with heteroatoms at the zigzag edges for improved stability and NLO properties. Those nanomaterials were further functionalized with electron-donating and electron-withdrawing groups (NH2/NO2) to enhance the NLO responses, and the connection of those functional groups at the azulene ends play a determinant role in the enhancement of the NLO properties of those X-type nanoribbons, e.g., the static first hyperpolarizability (〈ß0〉) changes from -783.23 × 10-30 esu to -1421.98 × 10-30 esu. The mechanism of such an enhancement has been investigated. Through two-dimensional second order NLO spectra simulations, particularly besides the strong electro-optical Pockels effect and optical rectification responses, strong electronic sum frequency generations and difference frequency generations are observed in those GNRs. The strong second order NLO responses of those GNRs in the visible light region bring about potential applications of these carbon nanomaterials in nonlinear nanophotonic devices and biological nonlinear optics.

Effect of lncRNA-ATB/miR-651-3p/Yin Yang 1 pathway on trophoblast-endothelial cell interaction networks.

Our previous studies have confirmed that lncRNA-ATB may be involved in the pathogenesis of preeclampsia, however, it is uncertain whether lncRNA-ATB influence the interaction between trophoblast and endothelial cells, which is crucial to the uterine spiral artery remodelling. Scratch wound healing and transwell invasion assay were conducted to test the migration and invasion of trophoblast cells. Co-culture model was used to simulate the physiological environment in vivo. The expression levels of lncRNA-ATB were analyzed in placenta tissues from healthy pregnant women and preeclampsia patients. Subsequently, the binding site of lncRNA-ATB and miR-651-3p was verified using dual-luciferase reporter assay, and the rescue experiment was used to study the effects of these two on the biological function. The direct effects of miR-651-3p and Yin Yang 1 (YY1) were verified using similar methods. LncRNA-ATB was found to be down-regulated in the placenta of preeclampsia patients. LncRNA-ATB knockdown decreased trophoblast migration, invasion and colocalisation with human umbilical vein endothelial cells. MiR-651-3p was a direct target of lncRNA-ATB and they had opposite effects. Moreover, the expression of lncRNA-ATB and miR-651-3p in placental tissues was negatively correlated. MiR-651-3p has been confirmed to directly target the 3' untranslated region of YY1. The inhibitory effects of YY1 low expression on biological function was rescued by miR-651-3p depletion. Western blot analysis showed that lncRNA-ATB could regulate YY1 expression by sponging miR-651-3p. LncRNA-ATB functioned as a competitive endogenous RNA of miR-651-3p to regulate YY1 on progress of spiral artery remodelling.

Improved plant cytosine base editors with high editing activity, purity, and specificity.

Cytosine base editors (CBEs) are great additions to the expanding genome editing toolbox. To improve C-to-T base editing in plants, we first compared seven cytidine deaminases in the BE3-like configuration in rice. We found A3A/Y130F-CBE_V01 resulted in the highest C-to-T base editing efficiency in both rice and Arabidopsis. Furthermore, we demonstrated this A3A/Y130F cytidine deaminase could be used to improve iSpyMacCas9-mediated C-to-T base editing at A-rich PAMs. To showcase its applications, we first applied A3A/Y130F-CBE_V01 for multiplexed editing to generate microRNA-resistant mRNA transcripts as well as pre-mature stop codons in multiple seed trait genes. In addition, we harnessed A3A/Y130F-CBE_V01 for efficient artificial evolution of novel ALS and EPSPS alleles which conferred herbicide resistance in rice. To further improve C-to-T base editing, multiple CBE_V02, CBE_V03 and CBE_V04 systems were developed and tested in rice protoplasts. The CBE_V04 systems were found to have improved editing activity and purity with focal recruitment of more uracil DNA glycosylase inhibitors (UGIs) by the engineered single guide RNA 2.0 scaffold. Finally, we used whole-genome sequencing (WGS) to compare six CBE_V01 systems and four CBE_V04 systems for genome-wide off-target effects in rice. Different levels of cytidine deaminase-dependent and sgRNA-independent off-target effects were indeed revealed by WGS among edited lines by these CBE systems. We also investigated genome-wide sgRNA-dependent off-target effects by different CBEs in rice. This comprehensive study compared 21 different CBE systems, and benchmarked PmCDA1-CBE_V04 and A3A/Y130F-CBE_V04 as next-generation plant CBEs with high editing efficiency, purity, and specificity.

Two-dimensional two-photon absorptions and third-order nonlinear optical properties of Ih fullerenes and fullerene onions.

The third order static and dynamic nonlinear optical (NLO) responses of Ih symmetry fullerenes (C60, C240, and C540) and fullerene onions (C60@C240 and C60@C240@C540) are predicted using the ZINDO method and the sum-over-states model. The static second hyperpolarizability of Ih symmetry fullerenes increases exponentially with fullerene size [from 10.00 × 10-34 esu in C60 to 3266.74 × 10-34 esu ≈ γ0(C60) × 92.63 in C540]. The external fields of strong third order NLO responses of Ih symmetry fullerenes change from ultra-violet (C60) to the visible region (C540) as the fullerene size increases. The outer layer fullerene in the fullerene onions has dominant contributions to the third order NLO properties of the fullerene onions, and the inter-shell charge-transfer excitations have conspicuous contributions to the third order NLO properties. The two-dimensional two-photon absorption spectra of C60 and C240 show that those fullerenes have strong two-photon absorptions in the visible region with short wavelength and in the ultra-violet region.

RIP1 promotes proliferation through G2/M checkpoint progression and mediates cisplatin-induced apoptosis and necroptosis in human ovarian cancer cells.

Receptor-interacting protein 1 (RIP1, also known as RIPK1) is not only a tumor-promoting factor in several cancers but also mediates either apoptosis or necroptosis in certain circumstances. In this study we investigated what role RIP1 plays in human ovarian cancer cells. We showed that knockout (KO) of RIP1 substantially suppressed cell proliferation, accompanied by the G2/M checkpoint arrest in two human ovarian cancer cell lines SKOV3 and A2780. On the other hand, RIP1 KO remarkably attenuated cisplatin-induced cytotoxicity, which was associated with reduction of the apoptosis markers PARP cleavage and the necroptosis marker phospho-MLKL. We found that RIP1 KO suppressed cisplatin-induced ROS accumulation in both SKOV3 and A2780 cells. ROS scavenger BHA, apoptosis inhibitor Z-VAD or necroptosis inhibitor NSA could effectively suppress cisplatin's cytotoxicity in the control cells, suggesting that ROS-mediated apoptosis and necroptosis were involved in cisplatin-induced cell death. In addition, blocking necroptosis with MLKL siRNA effectively attenuated cisplatin-induced cytotoxicity. In human ovarian cancer A2780 cell line xenograft nude mice, RIP1 KO not only significantly suppressed the tumor growth but also greatly attenuated cisplatin's anticancer activity. Our results demonstrate a dual role of RIP1 in human ovarian cancer: it acts as either a tumor-promoting factor to promote cancer cell proliferation or a tumor-suppressing factor to facilitate anticancer effects of chemotherapeutics such as cisplatin.

Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis.

BACKGROUND: CRISPR-Cas12a (formerly Cpf1) is an RNA-guided endonuclease with distinct features that have expanded genome editing capabilities. Cas12a-mediated genome editing is temperature sensitive in plants, but a lack of a comprehensive understanding on Cas12a temperature sensitivity in plant cells has hampered effective application of Cas12a nucleases in plant genome editing. RESULTS: We compared AsCas12a, FnCas12a, and LbCas12a for their editing efficiencies and non-homologous end joining (NHEJ) repair profiles at four different temperatures in rice. We found that AsCas12a is more sensitive to temperature and that it requires a temperature of over 28 °C for high activity. Each Cas12a nuclease exhibited distinct indel mutation profiles which were not affected by temperatures. For the first time, we successfully applied AsCas12a for generating rice mutants with high frequencies up to 93% among T0 lines. We next pursued editing in the dicot model plant Arabidopsis, for which Cas12a-based genome editing has not been previously demonstrated. While LbCas12a barely showed any editing activity at 22 °C, its editing activity was rescued by growing the transgenic plants at 29 °C. With an early high-temperature treatment regime, we successfully achieved germline editing at the two target genes, GL2 and TT4, in Arabidopsis transgenic lines. We then used high-temperature treatment to improve Cas12a-mediated genome editing in maize. By growing LbCas12a T0 maize lines at 28 °C, we obtained Cas12a-edited mutants at frequencies up to 100% in the T1 generation. Finally, we demonstrated DNA binding of Cas12a was not abolished at lower temperatures by using a dCas12a-SRDX-based transcriptional repression system in Arabidopsis. CONCLUSION: Our study demonstrates the use of high-temperature regimes to achieve high editing efficiencies with Cas12a systems in rice, Arabidopsis, and maize and sheds light on the mechanism of temperature sensitivity for Cas12a in plants.

Knockout of the OsNAC006 Transcription Factor Causes Drought and Heat Sensitivity in Rice.

Rice (Oryza sativa) responds to various abiotic stresses during growth. Plant-specific NAM, ATAF1/2, and CUC2 (NAC) transcription factors (TFs) play an important role in controlling numerous vital growth and developmental processes. To date, 170 NAC TFs have been reported in rice, but their roles remain largely unknown. Herein, we discovered that the TF OsNAC006 is constitutively expressed in rice, and regulated by H2O2, cold, heat, abscisic acid (ABA), indole-3-acetic acid (IAA), gibberellin (GA), NaCl, and polyethylene glycol (PEG) 6000 treatments. Furthermore, knockout of OsNAC006 using the CRISPR-Cas9 system resulted in drought and heat sensitivity. RNA sequencing (RNA-seq) transcriptome analysis revealed that OsNAC006 regulates the expression of genes mainly involved in response to stimuli, oxidoreductase activity, cofactor binding, and membrane-related pathways. Our findings elucidate the important role of OsNAC006 in drought responses, and provide valuable information for genetic manipulation to enhance stress tolerance in future plant breeding programs.

IL-25 protects against high-fat diet-induced hepatic steatosis in mice by inducing IL-25 and M2a macrophage production.

Interleukin (IL)-25 is a cytokine that has previously been shown to have a protective role against nonalcoholic fatty liver disease (NAFLD), which is associated with the induction of M2 macrophage differentiation. However, the direct relationships between IL-25 expression regulation, M2 induction and NAFLD remain unknown. In this study, we demonstrate that IL-25 promotes hepatic macrophage differentiation into M2a macrophages both in vivo and in vitro via the IL-13/STAT6 pathway. M2 macrophages that were differentiated in vitro were able to ameliorate high-fat diet HFD-induced hepatic steatosis. Furthermore, we found that IL-25 treatment, both in vitro and in vivo, promotes direct binding of STAT6 to the IL-25 gene promoter region. This binding of STAT6 in response to IL-25 treatment also resulted in the increase of IL-25 expression in hepatocytes. Together, these findings identify IL-25 as a protective factor against HFD-induced hepatic steatosis by inducing an increase of IL-25 expression in hepatocytes and through promotion of M2a macrophage production.

Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing.

CRISPR-Cas9 and Cas12a are two powerful genome editing systems. Expression of CRISPR in plants is typically achieved with a mixed dual promoter system, in which Cas protein is expressed by a Pol II promoter and a guide RNA is expressed by a species-specific Pol III promoter such as U6 or U3. To achieve coordinated expression and compact vector packaging, it is desirable to express both CRISPR components under a single Pol II promoter. Previously, we demonstrated a first-generation single transcript unit (STU)-Cas9 system, STU-Cas9-RZ, which is based on hammerhead ribozyme for processing single guide RNAs (sgRNAs). In this study, we developed two new STU-Cas9 systems and one STU-Cas12a system for applications in plants, collectively called the STU CRISPR 2.0 systems. We demonstrated these systems for genome editing in rice with both transient expression and stable transgenesis. The two STU-Cas9 2.0 systems process the sgRNAs with Csy4 ribonuclease and endogenous tRNA processing system respectively. Both STU-Cas9-Csy4 and STU-Cas9-tRNA systems showed more robust genome editing efficiencies than our first-generation STU-Cas9-RZ system and the conventional mixed dual promoter system. We further applied the STU-Cas9-tRNA system to compare two C to T base editing systems based on rAPOBEC1 and PmCDA1 cytidine deaminases. The results suggest STU-based PmCDA1 base editor system is highly efficient in rice. The STU-Cas12a system, based on Cas12a' self-processing of a CRISPR RNA (crRNA) array, was also developed and demonstrated for expression of a single crRNA and four crRNAs. Altogether, our STU CRISPR 2.0 systems further expanded the CRISPR toolbox for plant genome editing and other applications.

Multiplex QTL editing of grain-related genes improves yield in elite rice varieties.

KEY MESSAGE: Significant yield increase has been achieved by simultaneous introduction of three trait-related QTLs in three rice varieties with multiplex editing by CRISPR-Cas9. Using traditional breeding approaches to develop new elite rice varieties with high yield and superior quality is challenging. It usually requires introduction of multiple trait-related quantitative trait loci (QTLs) into an elite background through multiple rounds of crossing and selection. CRISPR-Cas9-based multiplex editing of QTLs represents a new breeding strategy that is straightforward and cost effective. To test this approach, we simultaneously targeted three yield-related QTLs for editing in three elite rice varieties, namely J809, L237 and CNXJ. The chosen yield-related QTL genes are OsGS3, OsGW2 and OsGn1a, which have been identified to negatively regulate the grain size, width and weight, and number, respectively. Our approach rapidly generated all seven combinations of single, double and triple mutants for the target genes in elite backgrounds. Detailed analysis of these mutants revealed differential contributions of QTL mutations to yield performance such as grain length, width, number and 1000-grain weight. Overall, the contributions are additive, resulting in 68 and 30% yield per panicle increase in triple mutants of J809 and L237, respectively. Our data hence demonstrates a promising genome editing approach for rapid breeding of QTLs in elite crop varieties.

Ectopic Expression of DREB Transcription Factor, AtDREB1A, Confers Tolerance to Drought in Transgenic Salvia miltiorrhiza.

Drought decreases crop productivity more than any other type of environmental stress. Transcription factors (TFs) play crucial roles in regulating plant abiotic stress responses. The Arabidopsis thaliana gene DREB1A/CBF3, encoding a stress-inducible TF, was introduced into Salvia miltiorrhiza Ectopic expression of AtDREB1A resulted in increased drought tolerance, and transgenic lines had higher relative water content and Chl content, and exhibited an increased photosynthetic rate when subjected to drought stress. AtDREB1A transgenic plants generally displayed lower malondialdehyde (MDA), but higher superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities under drought stress. In particular, plants with ectopic AtDREB1A expression under the control of the stress-induced RD29A promoter exhibited more tolerance to drought compared with p35S::AtDREB1A transgenic plants, without growth inhibition or phenotypic aberrations. Differential gene expression profiling of wild-type and pRD29A::AtDREB1A transgenic plants following drought stress revealed that the expression levels of various genes associated with the stress response, photosynthesis, signaling, carbohydrate metabolism and protein protection were substantially higher in transgenic plants. In addition, the amount of salvianolic acids and tanshinones was significantly elevated in AtDREB1A transgenic S. miltiorrhiza roots, and most of the genes in the related biosynthetic pathways were up-regulated. Together, these results demonstrated that inducing the expression of a TF can effectively regulate multiple genes in the stress response pathways and significantly improve the resistance of plants to abiotic stresses. Our results also suggest that genetic manipulation of a TF can improve production of valuable secondary metabolites by regulating genes in associated pathways.

A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation.

The relative ease, speed, and biological scope of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Protein9 (Cas9)-based reagents for genomic manipulations are revolutionizing virtually all areas of molecular biosciences, including functional genomics, genetics, applied biomedical research, and agricultural biotechnology. In plant systems, however, a number of hurdles currently exist that limit this technology from reaching its full potential. For example, significant plant molecular biology expertise and effort is still required to generate functional expression constructs that allow simultaneous editing, and especially transcriptional regulation, of multiple different genomic loci or multiplexing, which is a significant advantage of CRISPR/Cas9 versus other genome-editing systems. To streamline and facilitate rapid and wide-scale use of CRISPR/Cas9-based technologies for plant research, we developed and implemented a comprehensive molecular toolbox for multifaceted CRISPR/Cas9 applications in plants. This toolbox provides researchers with a protocol and reagents to quickly and efficiently assemble functional CRISPR/Cas9 transfer DNA constructs for monocots and dicots using Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing and transcriptional activation or repression of plant endogenous genes. We report the functionality and effectiveness of this toolbox in model plants such as tobacco (Nicotiana benthamiana), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa), demonstrating its utility for basic and applied plant research.