High performance TadA-8e derived cytosine and dual base editors with undetectable off-target effects in plants.

Cytosine base editors (CBEs) and adenine base editors (ABEs) enable precise C-to-T and A-to-G edits. Recently, ABE8e, derived from TadA-8e, enhances A-to-G edits in mammalian cells and plants. Interestingly, TadA-8e can also be evolved to confer C-to-T editing. This study compares engineered CBEs derived from TadA-8e in rice and tomato cells, identifying TadCBEa, TadCBEd, and TadCBEd_V106W as efficient CBEs with high purity and a narrow editing window. A dual base editor, TadDE, promotes simultaneous C-to-T and A-to-G editing. Multiplexed base editing with TadCBEa and TadDE is demonstrated in transgenic rice, with no off-target effects detected by whole genome and transcriptome sequencing, indicating high specificity. Finally, two crop engineering applications using TadDE are shown: introducing herbicide resistance alleles in OsALS and creating synonymous mutations in OsSPL14 to resist OsMIR156-mediated degradation. Together, this study presents TadA-8e derived CBEs and a dual base editor as valuable additions to the plant editing toolbox.

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.

MAPK20-mediated ATG6 phosphorylation is critical for pollen development in Solanum lycopersicum L.

In flowering plants, male gametogenesis is tightly regulated by numerous genes. Mitogen-activated protein kinase (MAPK) plays a critical role in plant development and stress response, while its role in plant reproductive development is largely unclear. The present study demonstrated MAPK20 phosphorylation of ATG6 to mediate pollen development and germination in tomato (Solanum lycopersicum L.). MAPK20 was preferentially expressed in the stamen of tomato, and mutation of MAPK20 resulted in abnormal pollen grains and inhibited pollen viability and germination. MAPK20 interaction with ATG6 mediated the formation of autophagosomes. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that ATG6 was phosphorylated by MAPK20 at Ser-265. Mutation of ATG6 in wild-type (WT) or in MAPK20 overexpression plants resulted in malformed and inviable pollens. Meanwhile, the number of autophagosomes in mapk20 and atg6 mutants was significantly lower than that of WT plants. Our results suggest that MAPK20-mediated ATG6 phosphorylation and autophagosome formation are critical for pollen development and germination.

Boosting genome editing in plants with single transcript unit surrogate reporter systems.

CRISPR-Cas-based genome editing holds immense promise for advancing plant genomics and crop enhancement. However, the challenge of low editing activity complicates the identification of editing events. In this study, we introduce multiple single transcript unit surrogate reporter (STU-SR) systems to enhance the selection of genome-edited plants. These systems use the same single guide RNAs designed for endogenous genes to edit reporter genes, establishing a direct link between reporter gene editing activity and that of endogenous genes. Various strategies are used to restore functional reporter genes after genome editing, including efficient single-strand annealing (SSA) for homologous recombination in STU-SR-SSA systems. STU-SR-base editor systems leverage base editing to reinstate the start codon, enriching C-to-T and A-to-G base editing events. Our results showcase the effectiveness of these STU-SR systems in enhancing genome editing events in the monocot rice, encompassing Cas9 nuclease-based targeted mutagenesis, cytosine base editing, and adenine base editing. The systems exhibit compatibility with Cas9 variants, such as the PAM-less SpRY, and are shown to boost genome editing in Brassica oleracea, a dicot vegetable crop. In summary, we have developed highly efficient and versatile STU-SR systems for enrichment of genome-edited plants.

Enhancing Energy Storage via Confining Sulfite Anions onto Iron Oxide/Poly(3,4-Ethylenedioxythiophene) Heterointerface.

Multiple oxidation-state metal oxide has presented a promising charge storage capability for aqueous supercapacitors (SCs); however, the ion insert/deinsert behavior in the bulk phase generally gives a sluggish reaction kinetic and considerable volume effect. Herein, iron oxide/poly(3,4-ethylenedioxythiophene) (Fe2O3/PEDOT) heterointerface was constructed and enabled boosted Faradaic pseudocapacitance by dual-ion-involved redox reactions in Na2SO3 electrolytes. The Fe2O3/PEDOT interface served as a "bridge" to couple electrode and anion SO32- and exhibited a strong force and stable bonding with SO32-, thus providing an additional Faradaic charge storage contribution for SCs. Significantly, the PEDOT-capsulated Fe2O3 nanorod array (Fe2O3@PEDOT) electrode presented a specific capacitance of 338 mF cm-2 at 1 mA cm-2 with 1 M Na2SO3 electrolyte, which was twice that of the pristine Fe2O3 nanorod electrode. The boosted interfaced Faradaic reaction of SO32- partially hindered the intercalation of Na+ in the Fe2O3 bulk phase, efficiently favoring the electrochemical stability.

Prophylactic alpha blockers fail to prevent postoperative urinary retention following orthopaedic procedures: evidence from a meta-analysis and trial sequential analysis of comparative studies.

Objective: The present systematic review and meta-analysis aimed to estimate the prophylactic effect of alpha blockers against postoperative urinary retention (POUR) in orthopaedic patients. Methods: PubMed, Embase, Web of Science and Cochrane Library databases were searched between 1 January 1990 and 1 March 2023. The studies reporting the preventive efficacy of alpha blockers on POUR after orthopaedic procedures were identified. The pooled rates of POUR in the Intervention group (patients receiving alpha blockers) and the Control group (patients not receiving alpha blockers) were estimated and compared. The risk ratios (RRs) were calculated using the random-effects model. Subgroup analysis was performed based on surgical type. Trial sequential analysis (TSA) was conducted to confirm the robustness of pooled results. Results: Seven studies containing 1,607 patients were identified. The rates of POUR were similar between the two groups (Intervention group: 126/748 [16.8%] VS. Control group: 168/859 [19.6%]; RR = 0.75; 95% confidence interval [CI] 0.51 to 1.09; p = 0.130; Heterogeneity: I2 = 67.1%; p = 0.006). No significant difference in the incidence of POUR was observed in either the Arthroplasty subgroup or Spine surgery subgroup. The result of TSA suggested that the total sample size of the existing evidence might be insufficient to draw conclusive results. Administrating alpha blockers was associated with a higher risk of complications (88/651 [13.5%] VS. 56/766 [7.3%]; RR = 1.73; 95% CI 1.27 to 2.37; p = 0.0005; Heterogeneity: I2 = 0%; p = 0.69). Conclusion: Prophylactic alpha blockers do not reduce the risk of POUR in orthopaedic procedures, and administrating these drugs was associated with a higher risk of complications. Systematic Review Registration: https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=409388.

Efficient plant genome engineering using a probiotic sourced CRISPR-Cas9 system.

Among CRISPR-Cas genome editing systems, Streptococcus pyogenes Cas9 (SpCas9), sourced from a human pathogen, is the most widely used. Here, through in silico data mining, we have established an efficient plant genome engineering system using CRISPR-Cas9 from probiotic Lactobacillus rhamnosus. We have confirmed the predicted 5'-NGAAA-3' PAM via a bacterial PAM depletion assay and showcased its exceptional editing efficiency in rice, wheat, tomato, and Larix cells, surpassing LbCas12a, SpCas9-NG, and SpRY when targeting the identical sequences. In stable rice lines, LrCas9 facilitates multiplexed gene knockout through coding sequence editing and achieves gene knockdown via targeted promoter deletion, demonstrating high specificity. We have also developed LrCas9-derived cytosine and adenine base editors, expanding base editing capabilities. Finally, by harnessing LrCas9's A/T-rich PAM targeting preference, we have created efficient CRISPR interference and activation systems in plants. Together, our work establishes CRISPR-LrCas9 as an efficient and user-friendly genome engineering tool for diverse applications in crops and beyond.

SULF1 Activates the VEGFR2/PI3K/AKT Pathway to Promote the Development of Cervical Cancer.

BACKGROUND AND PURPOSE: Sulfatase 1 (SULF1) can regulate the binding of numerous signaling molecules by removing 6-O-sulfate from heparan sulfate proteoglycans (HSPGs) to affect numerous physiological and pathological processes. Our research aimed to investigate the effect of the SULF1-mediated VEGFR2/PI3K/AKT signaling pathway on tumorigenesis and development of cervical cancer (CC). METHODS: The expression and prognostic values of SULF1 in patients with CC were analyzed through bioinformatics analysis, RT-PCR, immunohistochemistry, and western blot assays. The function and regulatory mechanism of SULF1 in proliferation, migration, and invasion of cervical cancer cells were examined through lentivirus transduction, CCK8, flow cytometry analysis, plate colony formation assay, scratch assay, transwell assay, western blot, VEGFR2 inhibitor (Ki8751), and mouse models. RESULTS: SULF1 expression was significantly upregulated in CC tissues, which was significantly associated with poor prognosis of patients with CC. In vitro, the upregulation of SULF1 expression in cervical cancer HeLa cells promoted cell proliferation, colony formation, migration, and invasion while inhibiting apoptosis. Conversely, the downregulation of SULF1 expression had the opposite effect. In vivo, the upregulation of SULF1 expression resulted in a significant increase in both tumor growth and angiogenesis, while its downregulation had the opposite effect. Furthermore, western blot detection and cell function rescue assay confirmed that the upregulation of SULF1 in HeLa cells promoted the tumorigenic behaviors of cancer cells by activating the VEGFR2/PI3K/AKT signaling pathway. CONCLUSION: SULF1 plays an oncogenic role in the tumorigenesis and development of CC, indicating its potential as a novel molecular target for gene-targeted therapy in patients with CC.

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.

An efficient CRISPR-Cas12a promoter editing system for crop improvement.

Promoter editing represents an innovative approach to introduce quantitative trait variation (QTV) in crops. However, an efficient promoter editing system for QTV needs to be established. Here we develop a CRISPR-Cas12a promoter editing (CAPE) system that combines a promoter key-region estimating model and an efficient CRISPR-Cas12a-based multiplexed or singular editing system. CAPE is benchmarked in rice to produce QTV continuums for grain starch content and size by targeting OsGBSS1 and OsGS3, respectively. We then apply CAPE for promoter editing of OsD18, a gene encoding GA3ox in the gibberellin biosynthesis pathway. The resulting lines carry a QTV continuum of semidwarfism without significantly compromising grain measures. Field trials demonstrated that the OsD18 promoter editing lines have the same yield performance and antilodging phenotype as the Green Revolution OsSD1 mutants in different genetic backgrounds. Hence, promoter editing of OsD18 generates a quantitative Green Revolution trait. Together, we demonstrate a CAPE-based promoter editing and tuning pipeline for efficient production of useful QTV continuum in crops.

Loss-function mutants of OsCKX gene family based on CRISPR-Cas systems revealed their diversified roles in rice.

Cytokinin (CTK) is an important plant hormone that promotes cell division, controls cell differentiation, and regulates a variety of plant growth and development processes. Cytokinin oxidase/dehydrogenase (CKX) is an irreversible cytokinin-degrading enzyme that affects plant growth and development by regulating the dynamic balance of CTKs synthesis and degradation. There are presumed 11 members of the CKX gene family in rice (Oryza sativa L.), but limited members have been reported. In this study, based on CRISPR-Cas9 and CRISPR-Cas12a genome-editing technology, we established a complete set of OsCKX1-OsCKX11 single-gene mutants, as well as double-gene and triple-gene mutants of different OsCKXs gene combinations with high similarity. The results revealed that CRISPR-Cas12a outperformed Cas9 to generate biallelic mutations, multi-gene mutants, and more diverse genotypes. And then, we found, except the reported OsCKX2, OsCKX4, OsCKX9 and OsCKX11, OsCKX5, OsCKX6, OsCKX7, and OsCKX8 also had significant effects on agronomic traits such as plant height, panicle size, grain size, and grain number per panicle in rice. In addition, the different loss-of-function of the OsCKX genes also changed the seed appearance quality and starch composition. Interestingly, by comparing different combinations of multi-gene mutants, we found significant functional redundancy among OsCKX gene members in the same phylogenetic clade. These data collectively reveal the diversified regulating capabilities of OsCKX genes in rice, and also provide the valuable reference for further rice molecular breeding.

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.

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.

Autophagic pathway contributes to low-nitrogen tolerance by optimizing nitrogen uptake and utilization in tomato.

Autophagy is a primary process involved in the degradation and reuse of redundant or damaged cytoplasmic components in eukaryotes. Autophagy has been demonstrated to facilitate nutrient recycling and remobilization by delivering intracellular materials to the vacuole for degradation in plants under nutrient starvation. However, the role of autophagy in nitrogen (N) uptake and utilization remains unknown. Here, we report that the ATG6-dependent autophagic pathway regulates N utilization in tomato (Solanum lycopersicum) under low-nitrogen (LN) conditions. Autophagy-disrupted mutants exhibited weakened biomass production and N accumulation compared with wild-type (WT), while ATG6 overexpression promoted autophagy and biomass production under LN stress. The N content in atg6 mutants decreased while that in ATG6-overexpressing lines increased due to the control of N transporter gene expression in roots under LN conditions. Furthermore, ATG6-dependent autophagy enhanced N assimilation efficiency and protein production in leaves. Nitrate reductase and nitrite reductase activities and expression were compromised in atg6 mutants but were enhanced in ATG6-overexpressing plants under LN stress. Moreover, ATG6-dependent autophagy increased plant carbon fixation and photosynthetic capacity. The quantum yield of photosystem II, photosynthetic N use efficiency and photosynthetic protein accumulation were compromised in atg6 mutants but were restored in ATG6-overexpressing plants. A WT scion grafted onto atg6 mutant rootstock and an atg6 scion grafted onto WT rootstock both exhibited inhibited LN-induced autophagy and N uptake and utilization. Thus, ATG6-dependent autophagy regulates not only N uptake and utilization as well as carbon assimilation but also nutrient recycling and remobilization in tomato plants experiencing LN stress.

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.

Functions of Redox Signaling in Pollen Development and Stress Response.

Cellular redox homeostasis is crucial for normal plant growth and development. Each developmental stage of plants has a specific redox mode and is maintained by various environmental cues, oxidants, and antioxidants. Reactive oxygen species (ROS) and reactive nitrogen species are the chief oxidants in plant cells and participate in cell signal transduction and redox balance. The production and removal of oxidants are in a dynamic balance, which is necessary for plant growth. Especially during reproductive development, pollen development depends on ROS-mediated tapetal programmed cell death to provide nutrients and other essential substances. The deviation of the redox state in any period will lead to microspore abortion and pollen sterility. Meanwhile, pollens are highly sensitive to environmental stress, in particular to cell oxidative burst due to its peculiar structure and function. In this regard, plants have evolved a series of complex mechanisms to deal with redox imbalance and oxidative stress damage. This review summarizes the functions of the main redox components in different stages of pollen development, and highlights various redox protection mechanisms of pollen in response to environmental stimuli. In continuation, we also discuss the potential applications of plant growth regulators and antioxidants for improving pollen vigor and fertility in sustaining better agriculture practices.

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.

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.