The rapid development of high-throughput sequencing techniques has led biology into the big-data era.Data analyses using various bioinformatics tools rely on programming and command-line environments,which are challen...The rapid development of high-throughput sequencing techniques has led biology into the big-data era.Data analyses using various bioinformatics tools rely on programming and command-line environments,which are challenging and time-consuming for most wet-lab biologists.Here,we present TBtools(a Toolkit for Biologists integrating various biological data-handling tools),a stand-alone software with a userfriendly interface.The toolkit incorporates over 130 functions,which are designed to meet the increasing demand for big-data analyses,ranging from bulk sequence processing to interactive data visualization.A wide variety of graphs can be prepared in TBtools using a new plotting engine("JIGplot")developed to maximize their interactive ability;this engine allows quick point-and-click modification of almost every graphic feature.TBtools is platform-independent software that can be run under all operating systems with Java Runtime Environment 1.6 or newer.It is freely available to non-commercial users at https://github.com/CJ-Chen/TBtools/releases.展开更多
Genome editing tools such as the clustered regularly interspaced short palindromic repeat （CRISPR）-associated system （Cas） have been widely used to modify genes in model systems including animal zygotes and human ...Genome editing tools such as the clustered regularly interspaced short palindromic repeat （CRISPR）-associated system （Cas） have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear （3PN） zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene （HBB）. However, the efficiency of homologous recombination directed repair （HDR） of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene （HBD）, which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.展开更多
Single-molecule, real-time sequencing developed by Pacific BioSciences offers longer read lengths than the second-generation sequencing （SGS） technologies, making it well-suited for unsolved problems in genome, tran...Single-molecule, real-time sequencing developed by Pacific BioSciences offers longer read lengths than the second-generation sequencing （SGS） technologies, making it well-suited for unsolved problems in genome, transcriptome, and epigenetics research. The highly-contiguous de novo assemblies using PacBio sequencing can close gaps in current reference assemblies and characterize structural variation （SV） in personal genomes. With longer reads, we can sequence through extended repetitive regions and detect mutations, many of which are associated with dis- eases. Moreover, PacBio transcriptome sequencing is advantageous for the identification of gene isoforms and facilitates reliable discoveries of novel genes and novel isoforms of annotated genes, due to its ability to sequence full-length transcripts or fragments with significant lengths. Addition- ally, PacBio＇s sequencing technique provides information that is useful for the direct detection of base modifications, such as methylation. In addition to using PacBio sequencing alone, many hybrid sequencing strategies have been developed to make use of more accurate short reads in conjunction with PacBio long reads. In general, hybrid sequencing strategies are more affordable and scalable especially for small-size laboratories than using PacBio Sequencing alone. The advent of PacBio sequencing has made available much information that could not be obtained via SGS alone.展开更多
WRKYgenee encode transcription factors that are Involved In the regulation of various biological processes. These zinc-finger proteins, especially those members mediating stress responses, are uniquely expanded In pla...WRKYgenee encode transcription factors that are Involved In the regulation of various biological processes. These zinc-finger proteins, especially those members mediating stress responses, are uniquely expanded In plants. To facilitate the study of the evolutionary history and functions of this eupergene family, we performed an exhaustive search for WRKY genes using HMMER and a Hidden Markov Model that was specifically trained for rice. This work resulted In a comprehensive list of WRKYgene models In Oryza sativa L. eep. indica and L. eep. Japonica. Mapping of these genes to Individual chromosomes facilitated elimination of the redundant, leading to the Identification of 98 WRKYgenee In Japonica and 102 In indica rice. These genes were further categorized according to the number and structure of their zinc-finger domains. Based on a phylogenetlc tree of the conserved WRKY domains and the graphic display of WRKY loci on corresponding indica and Japonica chromosomes, we Identified possible WRKY gene duplications within, and losses between the two closely related rice subspecies. Also reviewed are the roles of WRKY genes In disease resistance and responses to salicylic acid and Jaemonlc acid, seed development and germination mediated by glbberelllns, other developmental processes Including senescence, and responses to ablotlc stresses and abeclelc acid In rice and other plants. The signaling pathways mediating WRKY gene expreeelon are also discussed.展开更多
Precise and straightforward methods to edit the plant genome are much needed for functional genomics and crop improvement. Recently, RNA-guided genome editing using bacterial Type II cluster regularly interspaced shor...Precise and straightforward methods to edit the plant genome are much needed for functional genomics and crop improvement. Recently, RNA-guided genome editing using bacterial Type II cluster regularly interspaced short palindromic repeats （CRISPR）-associated nuclease （Cas） is emerging as an efficient tool for genome editing in microbial and animal systems. Here, we report the genome editing and targeted gene mutation in plants via the CRISPR-Cas9 sys- tem. Three guide RNAs （gRNAs） with a 20-22-nt seed region were designed to pair with distinct rice genomic sites which are followed by the protospacer-adjacent motif （PAM）. The engineered gRNAs were shown to direct the Cas9 nuclease for precise cleavage at the desired sites and introduce mutation （insertion or deletion） by error-prone non-homologous end joining DNA repairing. By analyzing the RNA-guided genome-editing events, the mutation efficiency at these target sites was estimated to be 3-8%. In addition, the off-target effect of an engineered gRNA-Cas9 was found on an imper- fectly paired genomic site, but it had lower genome-editing efficiency than the perfectly matched site. Further analysis suggests that mismatch position between gRNA seed and target DNA is an important determinant of the gRNA-Cas9 tar- geting specificity, and specific gRNAs could be designed to target more than 90% of rice genes. Our results demonstrate that the CRISPR-Cas system can be exploited as a powerful tool for gene targeting and precise genome editing in plants.展开更多
Plants have acquired sophisticated stress response systems to adapt to changing environments. It is important to understand plants＇ stress response mechanisms in the effort to improve crop productivity under stressfu...Plants have acquired sophisticated stress response systems to adapt to changing environments. It is important to understand plants＇ stress response mechanisms in the effort to improve crop productivity under stressful conditions. The AP2/ERF transcription factors are known to regulate diverse processes of plant development and stress responses. In this study, the molecular characteristics and biological functions of AP2/ERFs in a variety of plant species were analyzed. AP2/ERFs, especially those in DREB and ERF subfamilies, are ideal candidates for crop improvement because their overexpression enhances tolerances to drought, salt, freezing, as well as resistances to multiple diseases in the transgenic plants. The comprehensive analysis of physiological functions is useful in elucidating the biological roles of AP2/ERF family genes in gene interaction, pathway regulation, and defense response under stress environments, which should provide new opportunities for the crop tolerance engineering.展开更多
Seventy-five previously known plant microRNAs (miRNAs) were classified into 14 families according to their gene sequence identity. A total of 18,694 plant expressed sequence tags (EST) were found in the GenBank EST da...Seventy-five previously known plant microRNAs (miRNAs) were classified into 14 families according to their gene sequence identity. A total of 18,694 plant expressed sequence tags (EST) were found in the GenBank EST databases by comparing all previously known Arabidopsis miRNAs to GenBank’s plant EST databases with BLAST algorithms. After removing the EST sequences with high numbers (more than 2) of mismatched nucleotides, a total of 812 EST contigs were identified. After predicting and scoring the RNA secondary structure of the 812 EST sequences using mFold software, 338 new potential miRNAs were identified in 60 plant species. miRNAs are widespread. Some microRNAs may highly conserve in the plant kingdom, and they may have the same ancestor in very early evolution. There is no nucleotide substitution in most miRNAs among many plant species. Some of the new identified potential miRNAs may be induced and regulated by environmental biotic and abiotic stresses. Some may be preferentially expressed in specific tissues, and are regulated by developmental switching. These findings suggest that EST analysis is a good alternative strategy for identifying new miRNA candidates, their targets, and other genes. A large number of miRNAs exist in different plant species and play important roles in plant developmental switching and plant responses to environmental abiotic and biotic stresses as well as signal transduction. Environmental stresses and developmental switching may be the signals for synthesis and regulation of miRNAs in plants. A model for miRNA induction and expression, and gene regulation by miRNA is hypothesized.展开更多
基金This work was funded by the National Key Research and Developmental Program of China(2018YFD1000104)This work is also supported by awards to R.X.,Y.H.,and H.C.from the National Key Research and Developmental Program of China(2017YFD0101702,2018YFD1000500,2019YFD1000500)+4 种基金the National Science Foundation of China(#31872063)the Special Support Program of Guangdong Province(2019TX05N193)the Key-Area Research and Development Program of Guangdong Province(2018B020202011)the Guangzhou Science and Technology Key Project(201804020063)Support to M.H.F.comes from the NSF Faculty Early Career Development Program(IOS-1942437).
文摘The rapid development of high-throughput sequencing techniques has led biology into the big-data era.Data analyses using various bioinformatics tools rely on programming and command-line environments,which are challenging and time-consuming for most wet-lab biologists.Here,we present TBtools(a Toolkit for Biologists integrating various biological data-handling tools),a stand-alone software with a userfriendly interface.The toolkit incorporates over 130 functions,which are designed to meet the increasing demand for big-data analyses,ranging from bulk sequence processing to interactive data visualization.A wide variety of graphs can be prepared in TBtools using a new plotting engine("JIGplot")developed to maximize their interactive ability;this engine allows quick point-and-click modification of almost every graphic feature.TBtools is platform-independent software that can be run under all operating systems with Java Runtime Environment 1.6 or newer.It is freely available to non-commercial users at https://github.com/CJ-Chen/TBtools/releases.
基金This study was supported by the National Basic Research Program （973 Program） （Nos. 2010CB945401 and 2012CB911201）, the National Natural Science Foundation of China （Grant Nos. 91019020, 81330055, and 31371508）.
文摘Genome editing tools such as the clustered regularly interspaced short palindromic repeat （CRISPR）-associated system （Cas） have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear （3PN） zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene （HBB）. However, the efficiency of homologous recombination directed repair （HDR） of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene （HBD）, which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.
基金supported by the institutional fund of the Department of Internal Medicine, University of Iowa, USA
文摘Single-molecule, real-time sequencing developed by Pacific BioSciences offers longer read lengths than the second-generation sequencing （SGS） technologies, making it well-suited for unsolved problems in genome, transcriptome, and epigenetics research. The highly-contiguous de novo assemblies using PacBio sequencing can close gaps in current reference assemblies and characterize structural variation （SV） in personal genomes. With longer reads, we can sequence through extended repetitive regions and detect mutations, many of which are associated with dis- eases. Moreover, PacBio transcriptome sequencing is advantageous for the identification of gene isoforms and facilitates reliable discoveries of novel genes and novel isoforms of annotated genes, due to its ability to sequence full-length transcripts or fragments with significant lengths. Addition- ally, PacBio＇s sequencing technique provides information that is useful for the direct detection of base modifications, such as methylation. In addition to using PacBio sequencing alone, many hybrid sequencing strategies have been developed to make use of more accurate short reads in conjunction with PacBio long reads. In general, hybrid sequencing strategies are more affordable and scalable especially for small-size laboratories than using PacBio Sequencing alone. The advent of PacBio sequencing has made available much information that could not be obtained via SGS alone.
基金Supported by the National Center for Research Resources （NCRR） （P20 RR 016464）, a component of the National Institutes of Health （NIH）, Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NCRR or NIH. Publication of this paper is supported by the National Natural Science Foundation of China （30624808）. Acknowledgements The authors thank two former members of our laboratory namely Drs Zhonglin Zhang and Zhen Xie, for stimulating discussions and their contributions in preparing Figure 4 and compiling information for the biological functions of WRKYgenes. Dr Zhang is currently a postdoctoral fellow in Duke and Dr Xie is a postdoctoral fellow in Harvard.
文摘WRKYgenee encode transcription factors that are Involved In the regulation of various biological processes. These zinc-finger proteins, especially those members mediating stress responses, are uniquely expanded In plants. To facilitate the study of the evolutionary history and functions of this eupergene family, we performed an exhaustive search for WRKY genes using HMMER and a Hidden Markov Model that was specifically trained for rice. This work resulted In a comprehensive list of WRKYgene models In Oryza sativa L. eep. indica and L. eep. Japonica. Mapping of these genes to Individual chromosomes facilitated elimination of the redundant, leading to the Identification of 98 WRKYgenee In Japonica and 102 In indica rice. These genes were further categorized according to the number and structure of their zinc-finger domains. Based on a phylogenetlc tree of the conserved WRKY domains and the graphic display of WRKY loci on corresponding indica and Japonica chromosomes, we Identified possible WRKY gene duplications within, and losses between the two closely related rice subspecies. Also reviewed are the roles of WRKY genes In disease resistance and responses to salicylic acid and Jaemonlc acid, seed development and germination mediated by glbberelllns, other developmental processes Including senescence, and responses to ablotlc stresses and abeclelc acid In rice and other plants. The signaling pathways mediating WRKY gene expreeelon are also discussed.
文摘Precise and straightforward methods to edit the plant genome are much needed for functional genomics and crop improvement. Recently, RNA-guided genome editing using bacterial Type II cluster regularly interspaced short palindromic repeats （CRISPR）-associated nuclease （Cas） is emerging as an efficient tool for genome editing in microbial and animal systems. Here, we report the genome editing and targeted gene mutation in plants via the CRISPR-Cas9 sys- tem. Three guide RNAs （gRNAs） with a 20-22-nt seed region were designed to pair with distinct rice genomic sites which are followed by the protospacer-adjacent motif （PAM）. The engineered gRNAs were shown to direct the Cas9 nuclease for precise cleavage at the desired sites and introduce mutation （insertion or deletion） by error-prone non-homologous end joining DNA repairing. By analyzing the RNA-guided genome-editing events, the mutation efficiency at these target sites was estimated to be 3-8%. In addition, the off-target effect of an engineered gRNA-Cas9 was found on an imper- fectly paired genomic site, but it had lower genome-editing efficiency than the perfectly matched site. Further analysis suggests that mismatch position between gRNA seed and target DNA is an important determinant of the gRNA-Cas9 tar- geting specificity, and specific gRNAs could be designed to target more than 90% of rice genes. Our results demonstrate that the CRISPR-Cas system can be exploited as a powerful tool for gene targeting and precise genome editing in plants.
基金supported by the National Transgenic Key Project of MOA(201 1ZX08002-002 and 2009ZX08009-083B)
文摘Plants have acquired sophisticated stress response systems to adapt to changing environments. It is important to understand plants＇ stress response mechanisms in the effort to improve crop productivity under stressful conditions. The AP2/ERF transcription factors are known to regulate diverse processes of plant development and stress responses. In this study, the molecular characteristics and biological functions of AP2/ERFs in a variety of plant species were analyzed. AP2/ERFs, especially those in DREB and ERF subfamilies, are ideal candidates for crop improvement because their overexpression enhances tolerances to drought, salt, freezing, as well as resistances to multiple diseases in the transgenic plants. The comprehensive analysis of physiological functions is useful in elucidating the biological roles of AP2/ERF family genes in gene interaction, pathway regulation, and defense response under stress environments, which should provide new opportunities for the crop tolerance engineering.
文摘Seventy-five previously known plant microRNAs (miRNAs) were classified into 14 families according to their gene sequence identity. A total of 18,694 plant expressed sequence tags (EST) were found in the GenBank EST databases by comparing all previously known Arabidopsis miRNAs to GenBank’s plant EST databases with BLAST algorithms. After removing the EST sequences with high numbers (more than 2) of mismatched nucleotides, a total of 812 EST contigs were identified. After predicting and scoring the RNA secondary structure of the 812 EST sequences using mFold software, 338 new potential miRNAs were identified in 60 plant species. miRNAs are widespread. Some microRNAs may highly conserve in the plant kingdom, and they may have the same ancestor in very early evolution. There is no nucleotide substitution in most miRNAs among many plant species. Some of the new identified potential miRNAs may be induced and regulated by environmental biotic and abiotic stresses. Some may be preferentially expressed in specific tissues, and are regulated by developmental switching. These findings suggest that EST analysis is a good alternative strategy for identifying new miRNA candidates, their targets, and other genes. A large number of miRNAs exist in different plant species and play important roles in plant developmental switching and plant responses to environmental abiotic and biotic stresses as well as signal transduction. Environmental stresses and developmental switching may be the signals for synthesis and regulation of miRNAs in plants. A model for miRNA induction and expression, and gene regulation by miRNA is hypothesized.
