Seasonal shifts in assembly dynamics of phytoplankton communities in a humans-affected river in NE China

Identifying seasonal shift in phytoplankton community is essential for understanding the significance of eutrophication and finding biological indicators of ecological health of a lotic system.Phytoplankton communities,as well as the seasonal changes in the Ashi River Basin(ASRB)of Heilongjiang Province were investigated from April 2018 to January 2019.A survey in April(spring),July(summer),October(autumn),and January(winter)at 16 sampling sites was conducted.The composition,abundance,and biodiversity indices of phytoplankton were studied and 127 taxa of phytoplankton were identified.Among them,Bacillariophyta dominated the phytoplankton communities in the whole year.There were significant spatio-temporal changes in the structures of the phytoplankton communities during the study period.Trophic state index(TSI)show that the nutritional status of the ASRB was at mesotrophic-middle eutrophic levels.Redundancy analysis(RDA)revealed that total nitrogen(TN),water temperature(WT),oxidation reduction potential(ORP),pH,and dissolved oxygen(DO)were the critical factors in the dynamic phytoplankton community structure.The multivariate regression tree(MRT)analysis showed that Chlamydomonas microsphaerella Pascher et Jahoda,Melosira granulata(Ehrenberg)Ralfs,Merismopedia tenuissima Lemmermann,and Asterionella formosa Hassall were valuable indicators in the determination of water quality in ASRB.Our findings provide a scientific basis for water quality protection and management at basin scale.

A dual-function selection system enables positive selection of multigene CRISPR mutants and negative selection of Cas9-free progeny in Arabidopsis

The CRISPR/Cas9 technology revolutionizes targeted gene knockout in diverse organisms including plants.However,screening edited alleles,particularly those with multiplex editing,from herbicide-or antibiotic-resistant transgenic plants and segregating out the Cas9 transgene represent two laborious processes.Current solutions to facilitate these processes rely on different selection markers.Here,by taking advantage of the opposite functions of a D-amino acid oxidase(DAO)in detoxifying D-serine and in metabolizing non-toxic D-valine to a cytotoxic product,we develop a DAO-based selection system that simultaneously enables the enrichment of multigene edited alleles and elimination of Cas9-containing progeny in Arabidopsis thaliana.Among five DAOs tested in Escherichia coli,the one encoded by Trigonopsis variabilis(TvDAO)could confer slightly stronger D-serine resistance than other homologs.Transgenic expression of TvDAO in Arabidopsis allowed a clear distinction between transgenic and nontransgenic plants in both D-serine-conditioned positive selection and D-valine-conditioned negative selection.As a proof of concept,we combined CRISPR-induced single-strand annealing repair of a dead TvDAO with D-serine-based positive selection to help identify transgenic plants with multiplex editing,where D-serine-resistant plants exhibited considerably higher co-editing frequencies at three endogenous target genes than those selected by hygromycin.Subsequently,D-valine-based negative selection successfully removed Cas9 and TvDAO transgenes from the survival offspring carrying inherited mutations.Collectively,this work provides a novel strategy to ease CRISPR mutant identification and Cas9 transgene elimination using a single selection marker,which promises more efficient and simplified multiplex CRISPR editing in plants.

气化炉激冷室中多相流动过程模拟与结构优化

气流床煤气化技术是煤炭清洁利用的重要手段之一,气化炉作为气流床煤气化的核心设备,通常分为燃烧室和激冷室两部分。其中,下降管-上升管结构的激冷室在煤气化装置中广泛应用。本工作采用数值模拟的方法对激冷室中的气液两相流动进行研究,分析了激冷室中出现合成气从黑水管串气问题的原因,并针对串气问题提出了三种激冷室结构改进方案:延长黑水出水口(结构A);向下延长上升管(结构B);靠近黑水出口管方向,在上升管上增加挡板(结构C)。模拟结果表明三种改进方案均能够避免串气问题。此外,还对不同结构激冷室黑水池中液固两相流进行了数值模拟,研究激冷室结构对不同粒径灰渣颗粒带出率的影响,结果表明,结构C的激冷室灰渣带出率最小,其次为结构A,这两种结构方案适用于对滤饼无回收利用的情况;相比原始结构,结构B对粒径≤20μm的颗粒带出率较高,大颗粒带出率降低,更适合对滤饼中残碳回收利用的情况。

Multiplex and optimization of dCas9-TV-mediated gene activation in plants

Synthetic gene activators consisting of nucleasedead Cas9(dCas9)for single-guide RNA(sgRNA)-directed promoter binding and a transcriptional activation domain(TAD)represent new tools for gene activation from endogenous genomic locus in basic and applied plant research.However,multiplex gene coactivation by d Cas9-TADs has not been demonstrated in whole plants.There is also room to optimize the performance of these tools.Here,we report that our previously developed gene activator,dCas9-TV,could simultaneously upregulate OsGW7 and OsER1 in rice by up to 3,738 fold,with one sg RNA targeting to each promoter.The gene coactivation could persist to at least the fourth generation.Astonishingly,thepolycistronictRNA-sgRNAexpression under the maize ubiquitin promoter,a Pol II promoter,could cause enormous activation of these genes by up to>40,000-fold in rice.Moreover,the yeast GCN4 coiled coil-mediated dCas9-TV dimerization appeared to be promising for enhancing gene activation.Finally,we successfully introduced a self-amplification loop for dCas9-TV expression in Arabidopsis to promote the transcriptional upregulation of AtFLS2,a previously characterized dCas9-TV-refractory gene with considerable basal expression.Collectively,this work illustrates the robustness of dCas9-TV in multigene coactivation and provides broadly useful strategies for boosting transcriptional activation efficacy of dCas9-TADs in plants.

A novel RNA-guided RNA-targeting CRISPR tool

Clustered regularly interspaced short palindromic repeats （CRISPR）/CRISPR-associated protein （Cas） technology has emerged as a programmable RNA-guided DNA-targeting tool for sequence-specific genome editing in diverse organ- isms. The most frequently used CRISPR/Cas system, which is based on the bacterium Streptococcus pyogenes Cas9 （SpCas9） endonuclease, has been repurposed into a suite of versatile tools for numerous DNA-manipulating activities beyond genome editing （Jiang and Marraffini, 2015）. How- ever, progress in converting the CRISPR/SpCas9 system into a sequence-specific RNA-manipulating platform has been limited. A recent study published in Science by Abudayyeh and colleagues （Abudayyeh et al., 2016） has identified Leptotrichia shahii C2c2 （LshC2c2） to be a novel programmable RNA-targeting endoribonuclease with great potential to reshape the landscape of future RNA research.

New cytosine base editor for plant genome editing

A large number of beneficial agronomic traits in crops are associated with single nucleotide polymorphisms(SNPs)or point mutations(Jiao et al.,2010;Li et al.,2017;Ma et al.,2015).In the past,site-specific point mutations in a target gene can only be achieved through the CRISPR/Cas9 mediated gene replacement via the homology-directed repair(HDR).However,the intrinsically low HDR activity in plant cells and the lack of efficient way to supply abundant HDR templates in plant nucleus have greatly limited the success rate of gene replacement in plants(Ran et al.,2017).In addition,DNA double-strand breaks(DSBs)generated by the Cas9 nuclease prior to HDR may lead to complicated and unexpected genome disturbance(Kosicki et al.,2018).

The working dead:repurposing inactive CRISPR-associated nucleases as programmable transcriptional regulators in plants

Targeted gene manipulation is highly desirable for fundamental plant research,plant synthetic biology,and molecular breeding.The clustered regularly interspaced short palindromic repeats-associated(Cas)nuclease is a revolutionary tool for genome editing,and has received snowballing popularity for gene knockout applications in diverse organisms including plants.Recently,the nuclease-dead Cas(dCas)proteins have been repurposed as programmable transcriptional regulators through translational fusion with portable transcriptional repression or activation domains,which has paved new ways for flexible and multiplex control over the activities of target genes of interest without the need to generate DNA lesions.Here,we review the most important breakthroughs of dCas transcriptional regulators in non-plant organisms and recent accomplishments of this growing field in plants.We also provide perspectives on future development directions of dCas transcriptional regulators in plant research in hope to stimulate their quick evolution and broad applications.

Targeted Gene Manipulation in Plants Using the CRISPR/Cas Technology

The CRISPR/Cas technology is emerging as a revolutionary genome editing tool in diverse organisms including plants,and has quickly evolved into a suite of versatile tools for sequence-specific gene manipulations beyond genome editing.Here,we review the most recent applications of the CRISPR/Cas toolkit in plants and also discuss key factors for improving CRISPR/Cas performance and strategies for reducing the off-target effects.Novel technical breakthroughs in mammalian research regarding the CRISPR/Cas toolkit will also be incorporated into this review in hope to stimulate prospective users from the plant research community to fully explore the potential of these technologies.