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Efficient and multiplex gene upregulation in plants through CRISPR-Cas-mediated knockin of enhancers

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MOLECULAR PLANT 2024年第9期17卷 1472-1483页

作者： Yao, Qi shen, rundong Shao, Yang Tian, Yifu Han, Peijin Zhang, Xuening Zhu, Jian-Kang Lu, Yuming Shanghai Jiao Tong Univ Shanghai Collaborat Innovat Ctr Agriseeds Joint Ctr Single Cell Biol Sch Agr & Biol Shanghai 200240 Peoples R China Chinese Acad Sci Shanghai Ctr Plant Stress Biol Ctr Excellence Mol Plant Sci Shanghai 201602 Peoples R China Univ Chinese Acad Sci Beijing Peoples R China Huazhong Agr Univ Coll Plant Sci & Technol Wuhan 430070 Peoples R China Southern Univ Sci & Technol Inst Adv Biotechnol Shenzhen 518055 Peoples R China Southern Univ Sci & Technol Sch Med Shenzhen 518055 Peoples R China

Gene upregulation through genome editing is important for plant research and breeding. Targeted insertion of short transcriptional enhancers (STEs) into gene promoters may offer a universal solution akin to transgene-mediated overexpression while avoiding the drawbacks associated with transgenesis. Here, we introduce an "in locus activation"technique in rice that leverages well-characterized STEs for refined, heritable, and multiplexed gene upregulation. To address the scarcity of potent enhancers, we developed a large-scale mining approach and discovered a suite of STEs that are capable of enhancing gene expression in rice protoplasts. The in locus integration of these STEs into eight rice genes resulted in substantial transcriptional upregulation in the edited plants, with up to 869.1-fold increases in their transcript levels. Employing a variety of STEs, we achieved delicate control of gene expression, enabling the fine-tuning of key phenotypic traits such as plant height. Our approach also enabled efficient multiplexed gene upregulation, with up to four genes activated simultaneously, significantly enhancing the nicotinamide mononucleotide metabolic pathway. Importantly, heritability studies from the T0 to T3 generations confirmed the stable and heritable nature of STE-driven gene activation. Collectively, our work demonstrates that coupled with STE mining, leveraging genome editing for in locus activation and gene upregulation holds great promise to be widely adopted in fundamental plant research and crop breeding.

关键词： genome editing short transcriptional enhancers gene upregulation crop breeding QUANTITATIVE TRAIT VARIATION CROP IMPROVEMENT GENOME

High-throughput genome editing in rice with a virus-based surrogate system

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Journal of Integrative Plant Biology 2023年第3期65卷 646-655页

作者： Yifu Tian Dating Zhong Xinbo Li rundong shen Han Han Yuqin Dai Qi Yao Xuening Zhang Qi Deng Xuesong Cao Jian-Kang Zhu Yuming Lu Shanghai Center for Plant Stress Biology Center for Excellence in Molecular Plant SciencesChinese Academy of SciencesShanghai 201602China Center for Advanced Bioindustry Technologies Institute of Crop SciencesChinese Academy of Agricultural SciencesBeiing 100081China Hainan Yazhou Bay Seed Lab Sanya 572024China Shanghai Collaborative Innovation Center of Agri Seeds Joint Center for Single Cell BiologySchool of Agriculture and BiologyShanghai Jiao Tong UniversityShanghai 200240China Institute of Advanced Biotechnology School of Life SciencesSouthern University of Science and TechnologyShenzhen 518055China

With the widespread use of clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated nuclease(Cas) technologies in plants, large-scale genome editing is increasingly needed. Here, we developed a geminivirus-mediated surrogate system, called Wheat Dwarf Virus-Gate(WDV-surrogate), to facilitate high-throughput genome ***-Gate has two parts: one is the recipient callus from a transgenic rice line expressing Cas9 and a mutated hygromycin-resistant gene(HygM) for surrogate selection;the other is a WDV-based construct expressing two single guide RNAs(sgRNAs) targeting HygM and a gene of interest, respectively. We evaluated WDV-Gate on six rice loci by producing a total of 874 T_0 plants. Compared with the conventional method, the WDV-Gate system, which was characterized by a transient and high level of sgRNA expression, significantly increased editing frequency(66.8% vs. 90.1%), plantlet regeneration efficiency(2.31-fold increase), and numbers of homozygous-edited plants(36.3%vs. 70.7%). Large-scale editing using pooled sg RNAs targeting the SLR1 gene resulted in a high editing frequency of 94.4%, further demonstrating its feasibility. We also tested WDVGate on sequence knock-in for protein *** co-delivering a chemically modified donor DNA with the WDV-Gate plasmid, 3xFLAG peptides were successfully fused to three loci with an efficiency of up to 13%. Thus, by combining transiently expressed sgRNAs and a surrogate selection system, WDV-Gate could be useful for high-throughput gene knock-out and sequence knock-in.

关键词： genome editing high-throughput protein tagging rice

Design of high-performance and sustainable Co-free Ni-rich cathodes for next-generation lithium-ion batteries

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SusMat 2024年第1期4卷 48-71页

作者： Hao Ge Zhiwen shen Yanhong Wang Zhijia Sun Xiaoman Cao Chaoyue Wang Xinyue Fan Jinsong Bai rundong Li Tianhua Yang Gang Wu School of Energy and Environment Shenyang Aerospace UniversityShenyangP.R.China shenyang Aircraft Airworthiness Certification Center of CAAC ShenyangP.R.China College of Chemistry and Materials Engineering Bohai UniversityJinzhouP.R.China Liaoning General Aviation Academy ShenyangP.R.China Department of Chemical and Biological Engineering University at BuffaloState University of New YorkBuffaloNew YorkUSA

Great attention has been given to high-performance and inexpensive lithiumion batteries(LIBs)in response to the ever-increasing demand for the explosive growth of electric vehicles(EVs).High-performance and low-cost Co-freeNi-rich layered cathodes are considered one of the most favorable candidates for nextgeneration LIBs because the current supply chain of EVs relies heavily on scarce and expensive ***,we review the recent research progress on Co-free Nirich layered cathodes,emphasizing on analyzing the necessity of replacing Co and the popular improvment *** current advancements in the design strategies of Co-free Ni-rich layered cathodes are summarized in *** considerable improvements achieved so far,the main technical challenges contributing to the deterioration of Co-free Ni-rich cathodes such as detrimental phase transitions,crack formation,and severe interfacial side reactions,are difficult to resolve by a single *** cooperation of multiple modification strategies is expected to accelerate the industrialization of Co-free Ni-rich layered cathodes,and the corresponding synergistic mechanisms urgently need to be *** effects will be aroused to explore high-performance Co-free Ni-rich layered cathodes to promote the sustainable development of LIBs.

关键词： Co-free cathodes electrochemical performance lithium-ion batteries modification strategies Ni-rich layered cathodes

Highly efficient recovery of waste LiNi_(x)Co_(y)Mn_(z)O_(2) cathode materials using a process involving pyrometallurgy and hydrometallurgy

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Frontiers of Environmental Science & Engineering 2024年第2期18卷 149-160页

作者： Tianwei Zhang Juanye Dao Jinsong Wang Yuzhong Guo rundong Wan Chengping Li Xian Zhou Zhengfu Zhang Faculty of Materials Science and Engineering Kunming University of Science and TechnologyKunming 650093China

Substantial environmental and economic benefits can be achieved by recycling used lithium-ion batteries. Hydrometallurgy is often employed to recover waste LiNi_(x)Co_(y)Mn_(z)O_(2) cathode materials. As Ni, Co and Mn are transition metals, they exhibit similar properties;therefore, separating them is difficult. Thus, most researchers have focused on leaching processes, while minimal attention has been devoted to the separation of valuable metals from waste LiNi_(x)Co_(y)Mn_(z)O_(2) cathode materials. Herein, we propose an environment-friendly, gentle process involving the usage of pyrometallurgy and hydrometallurgy to gradually leach valuable metals and effectively separate them. Interestingly, Li is recovered through a reduction roasting and water leaching process using natural graphite powder, Ni and Co are recovered through ammonia leaching and extraction processes and Mn is recovered through acid leaching and evaporation–crystallization processes. Results show that ~87% Li, 97.01% Co, 97.08% Ni and 99% Mn can be leached using water, ammonia and acid leaching processes. The result obtained using the response surface methodology shows that the concentration of (NH4)2SO3 is a notable factor affecting the leaching of transition metals. Under optimal conditions, ~97.01% Co, 97.08% Ni and 0.64% Mn can be leached out. The decomposition of LiNi_(x)Co_(y)Mn_(z)O_(2) is a two-step process. This study provides valuable insights to develop an environment-friendly, gentle leaching process for efficiently recycling valuable metals, which is vital for the lithium-ion battery recycling industry.

关键词： Waste LiNixCoyMnzO2 cathode materials Leaching Effective separation Series process

Drivers of spatial structure in thinned forests

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Forest Ecosystems 2024年第2期11卷 202-213页

作者： Zichun Wang Yaoxiang Li Guangyu Wang Zheyu Zhang Ya Chen Xiaoli Liu rundong Peng College of Mechanical and Electrical Engineering Northeast Forestry UniversityHarbin150040China Department of Forest Resources Management University of British ColumbiaVancouverCanada

Background:As is widely known,an increasing number of forest areas were managed to preserve and enhance the health of forest ***,previous research on forest management has often overlooked the importance of ***:Our objectives were to define the direction of structure-based forest ***,we investigated the relationships between forest structure and the regeneration,growth,and mortality of trees under different thinning ***,the drivers of forest structural change were ***:On the basis of 92 sites selected from northeastern China,with different recovery time (from 1 to 15years) and different thinning intensities (0–59.9%) since the last *** component analysis (PCA)identified relationships among factors determining forest spatial *** structural equation model (SEM)was used to analyze the driving factors behind the changes in forest spatial structure after ***:Light thinning (0–20%trees removed) promoted forest regeneration,and heavy thinning (over 35% of trees removed) facilitated forest ***,only moderate thinning (20%–35%trees removed) created a reasonable spatial *** dead trees were clustered,and they were hardly affected by thinning ***,thinning intensity,recovery time,and altitude indirectly improve the spatial structure of the forest by influencing diameter at breast height (DBH) and canopy ***:Creating larger DBH and canopy area through thinning will promote the formation of complex forest structures,which cultivates healthy and stable forests.

关键词： Thinning Natural secondary forest Spatial structure Dynamic changes Growth factors Structural equation modeling

White Radish polyphenols improve diet-induced obesity by regulating the metabolism of bile acids via gut-liver axis

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Food Science and Human Wellness 2024年

作者： Xiong Geng Weina Tian Miaomiao Zhuang rundong Wang Wenting Ma Ran Lin Huayan Shang Zhangfu Yu Ronghu shen Jianrong Li College of Food Science and Engineering， Bohai University Hangzhou Xiaoshan Agricultural Development CO.， Ltd

Natural plant or functional food-derived biologically active ingredients have recently raised considerable interest in their antiobesity effect. However， the antiobesity role of polyphenols of white radish roots remains unknown. This study examined the therapeutic efficacy and underlying mechanisms of polyphenols extract from white radish roots (RPE) on obese mice. RPE administration significantly decreased the weight gain in high-fat diet fed (HFD) mouse models. RPE intervention also increased serum glutathione transferase (GSH-Px) and superoxide dismutase (SOD) levels， and decreased malondialdehyde (MDA)， tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) levels in the HFD-fed mice. The intervention led to an escalation in the proportion of beneficial bacteria (Akkermansia， Clostridia)， and a corresponding decline in the fraction of harmful bacteria (Proteobacteria). According to liquid chromatography-mass spectrometry (LC-MS)-based gut metabolomics， the RPE intervention notably influenced several metabolic pathways， including those involved in protein digestion and absorption， central carbon metabolism related to cancer， and the biosynthesis of steroid hormones. Additionally， RPE treatment led to a reduction in the intestinal microbes possessing bile-salt hydrolase (BSH) activity， whereas increased the expression levels of the bile acid (BA) synthesis enzyme cholesterol 7α-hydroxylase (Cyp7a1). This led to increase hepatic production and fecal excretion of BAs， reduce hepatic cholesterol， and decrease lipogenesis. Treated HDF mice showed mitigation of hepatic steatosis and inflammation through gut-liver axis modulation by RPE interference. These findings together offer a significant understanding of the potential of RPE as an anti-obesity treatment and could assist in developing strategies for weight loss.

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Identification,characterization,and verification of miR399 target gene in grape

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Horticultural Plant Journal 2024年第1期10卷 91-102页

作者： Maosong Pei Hainan Liu Tonglu Wei Huiying Jin Yihe Yu Mengting Ma Xiaomeng Song rundong Dai Dalong Guo Henan Engineering Technology Research Center of Quality Regulation and Controlling of Horticultural Plants College of Horticulture and Plant ProtectionHenan University of Science and TechnologyLuoyangHenan 471023China

The microRNA miR399 plays an important role in phosphorus signal transduction pathways in ***,miR399 was shown to be closely associated with berry ripening in grape(Vitis vinifera).The objective of the present study was to elucidate the evolutionary characteristics of the miR399 gene family in grape and to verify the cleavage effect on the target *** miR399s were identified by miRNA sequencing and retrieval from the miRBase *** mature sequences and precursor sequences were subjected to phylogenetic analysis to reconstruct evolutionary trees,as well as secondary structure analysis of the precursor sequence,and prediction of target *** cisacting elements in the miR399 promoter were predicted and the cleavage effect of grape miR399b on its target genes was *** grape miR399 family comprised nine precursor sequences and nine mature *** precursor sequences formed a typical and stable stem—loop *** minimum folding free energy ranged from-55.70 kcal·mol^(-1)to-37.40 kcal·mol^(-1).Multiple sequence alignment revealed that the miR399 family was highly *** grape miR399 family was phylogenetically closely related to peach,apple,and citrus *** miR399s were predicted to target inorganic phosphate transporter 1—3,phospholipase D delta-like,and *** cleavage effect of grape miR399b on the target genes was verified by means of a dual-luciferase assay and 5’*** GUS staining showed that the promoter activity of miR399b was promoted by GA3treatment.

关键词： Grape miR399 Phosphorus metabolism Early ripening

Transient Characteristics and Accommodative Current Limiting Strategy for Bidirectional Interlinking Converters in Hybrid AC/DC Microgrids

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CSEE Journal of Power and Energy Systems 2024年第4期10卷 1575-1588页

作者： Xia shen Zhikang Shuai Wen Huang Chao shen Yang shen Z.John shen College of Electrical and Information Engineering Hunan UniversityChangsha 410082China Department of Electrical and Computer Engineering Illinois Institute of TechnologyChicagoIL 60616 USA

Bidirectional interlinking converter(BIC)is the core equipment in a hybrid AC/DC microgrid connected between AC and DC ***,the variety of control modes and flexible bidirectional power flow complicate the influence of AC faults on BIC itself and on DC sub-grid,which potentially threaten both converter safety and system *** study first investigates AC fault influence on the BIC and DC bus voltage under different BIC control modes and different pre-fault operation states,by developing a mathematical model and equivalent sequence ***,based on the analysis results,a general accommodative current limiting strategy is proposed for BIC without limitations to specific mode or operation *** amplitude is predicted and constrained according to the critical requirements to protect the BIC and relieving the AC fault influence on the DC bus *** with conventional methods,potential current limit failure and distortions under asymmetric faults can also be ***,experiments verify feasibility of the proposed method.

关键词： Bidirectional interlinking converter current limiting fault characteristic hybrid AC/DC microgrid variation/oscillation of DC bus voltage

Rational Solutions of First Order Algebraic Ordinary Differential Equations

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Journal of Systems Science & Complexity 2024年第2期37卷 567-580页

作者： FENG Shuang shen Liyong School of Physical and Mathematical Sciences Nanjing Tech UniversityNanjing 211816China School of Mathematical Sciences University of Chinese Academy of SciencesBeijing 100049China

Let f(t,y,y')=∑ _(i=0)^(n )a_(i)(t,y)y'^(i)=0 be an irreducible first order ordinary differential equation with polynomial *** in 1998 proved that there exists a constant C such that every rational solution of f(t,y,y')=0 is of degree not greater than *** show that this degree bound C depends not only on the degrees of f in t,y,y' but also on the coefficients of f viewed as the polynomial in t,y,y'.In this paper,the authors show that if f satisfies deg(f,y)0,then the degree bound C only depends on the degrees of f in t,y,y',and furthermore we present an explicit expression for C in terms of the degrees of f in t,y,y'.

关键词： Degree bound first order AODE height rational solution