Agrobacterium-mediated delivery of CRISPR/Cas reagents for genome editing in plants enters an era of ternary vector systems

Lack of appropriate methods for delivery of genome-editing reagents is a major barrier to CRISPR/Cas-mediated genome editing in plants. Agrobacterium-mediated genetic transformation(AMGT) is the preferred method of CRISPR/Cas reagent delivery,and researchers have recently made great improvements to this process. In this article, we review the development of AMGT and AMGT-based delivery of CRISPR/Cas reagents. We give an overview of the development of AMGT vectors including binary vector, superbinary vector, dual binary vector, and ternary vector systems. We also review the progress in Agrobacterium genomics and Agrobacterium genetic engineering for optimal strains. We focus in particular on the ternary vector system and the resources we developed. In summary, it is our opinion that Agrobacterium-mediated CRISPR/Cas genome editing in plants is entering an era of ternary vector systems, which are often integrated with morphogenic regulators. The new vectors described in this article are available from Addgene and/or MolecularCloud for sharing with academic investigators for noncommercial research.     

Enzymes with noncanonical amino acids

Enzyme design and engineering strategies rely almost exclusively on nature's alphabet of twenty canonical amino acids. Recent years have seen the emergence of powerful genetic code expansion methods that allow hundreds of structurally diverse amino acids to be installed into proteins in a site-selective manner. Here, we will highlight how the availability of an expanded alphabet of amino acids has opened new avenues in enzyme engineering research. Genetically encoded noncanonical amino acids have provided new tools to probe complex enzyme mechanisms, improve biocatalyst activity and stability, and most ambitiously to design enzymes with new catalytic mechanisms that would be difficult to access within the constraints of the genetic code. We anticipate that the studies highlighted in this article, coupled with the continuing advancements in genetic code expansion technology, will promote the widespread use of noncanonical amino acids in biocatalysis research in the coming years.     

几种新型植物基因表达载体的构建方法

利用基因工程技术手段研究基因功能过程中,构建基因表达载体处于转基因植物的主导地位,采用合适的构建方法会使实验效果事半功倍。植物基因表达载体的构建方法除了传统构建法、Gateway技术、三段T-DNA法、一步克隆法等,还有近年来出现的几种新型的载体构建方法:基于竞争性连接原理快速构建小片段基因表达载体;MicroRNA前体PCR置换法适用于构建小分子RNA表达载体;重组融合PCR法特别适用于插入片段中含有较多限制性酶切位点的载体构建;利用In-Fusion试剂盒可以将任何目的片段插入一个线性化载体的某个区域;构建多片段复杂载体可采用不依赖序列和连接的克隆方法(Sequence and ligation-independent cloning,SLIC)法;Gibson等温拼接法;Golden Gate拼接法。本文将在总结分析前人工作的基础上,结合自己工作的体会和经验分析这7种新方法的特点,期望通过这几种新的方法给植物基因工程表达载体的构建提供新的思路。     

丝状真菌表面展示技术研究进展

丝状真菌表面展示技术是将表达的目的蛋白固定在丝状真菌细胞表面的一项新兴基因工程技术。丝状真菌具有极强的蛋白质分泌能力和良好的蛋白质翻译后加工能力,因而越来越多的丝状真菌表面展示技术得到开发和应用。本文就丝状真菌表面展示系统的研发和应用进展进行综述,并介绍与该系统构建密切相关的丝状真菌的细胞壁组成、锚定蛋白和遗传转化方法等技术。     

用于通路(Gateway)克隆技术的植物表达载体研究进展

通路(Gateway)克隆技术是根据λ噬菌体基因组和大肠杆菌基因组之间的位点专一性重组分子机制开发的一套分子克隆新技术.利用该技术LR反应构建目的基因的表达载体时不需要经过酶切和连接等繁琐而又费时的过程,因此,可以节省很多时间.为了扩大Gateway技术在植物基因工程领域的应用,最近有很多研究机构和研究小组开发了能用于组成型或诱导型表达目的基因、基因沉默、启动子分析、蛋白质亚细胞定位、蛋白质/蛋白质相互作用、多个DNA片段的模块化组装和DNA组片段功能验证等研究用的植物表达载体.该文对这些技术的研究进展进行了综述.     

双元Ti载体的发展

双元Ti载体是目前植物基因工程中最重要的植物转化载体。近年来双元Ti载体得到迅速的发展,新的载体不断出现。新发展的双元Ti载体结构优化,适用范围广,易于基因克隆操作,同时提高了植物转化的效率,更便于进行植物基因功能的研究。本文介绍了构建高容量载体、多基因载体、定点整合载体所采用的新策略。     

Metabolic engineering

Metabolic engineering has developed as a very powerful approach to optimising industrial fermentation processes through the introduction of directed genetic changes using recombinant DNA technology. Successful metabolic engineering starts with a careful analysis of cellular function; based on the results of this analysis, an improved strain is designed and subsequently constructed by genetic engineering. In recent years some very powerful tools have been developed, both for analysing cellular function and for introducing directed genetic changes. In this paper, some of these tools are reviewed and many examples of metabolic engineering are presented to illustrate the power of the technology. The examples are categorised according to the approach taken or the aim: (1) heterologous protein production, (2) extension of substrate range, (3) pathways leading to new products, (4) pathways for degradation of xenobiotics, (5) improvement of overall cellular physiology, (6) elimination or reduction of by-product formation, and (7) improvement of yield or productivity.     

Tools for genetic engineering in the amino acid-producing bacterium Corynebacterium glutamicum

During the last decades, the gram-positive soil bacterium Corynebacterium glutamicum has been shown to be a very versatile microorganism for the large-scale fermentative production of L-amino acids. Up to now, a vast amount of techniques and tools for genetic engineering and amplification of relevant structural genes have been developed. The objectives of this study are to summarize the published literature on tools for genetic engineering in C. glutamicum and to focus on new sophisticated and highly efficient methods in the fields of DNA transfer techniques, cloning vectors, integrative genetic tools, and antibiotic-free self-cloning. This repertoire of C. glutamicum methodology provides an experimental basis for efficient genetic analyses of the recently completed genome sequence.     

Yeast recombinase FLP functions effectively in human cells for construction of adenovirus vectors

We have recently developed a high-efficiency method of constructing adenovirus vectors based on Cre-mediated recombination between two plasmids co-transfected into 293 cells. The simplicity and efficiency of this method should greatly expedite the construction of most recombinant vectors. However, this system would not be suitable for constructing vectors bearing loxP sites elsewhere in the genome because of undesirable Cre-mediated vector rearrangements. To address this, we have developed a similar system using FLP-mediated site-specific recombination for the construction of adenovirus vectors.     

Genetic tools for <Emphasis Type="Italic">Sulfolobus</Emphasis> spp.: vectors and first applications

Sulfolobus species belong to the best-studied archaeal organisms but have lacked powerful genetic methods. Recently, there has been considerable progress in the field of Sulfolobus genetics. Urgently needed basic genetic tools, such as targeted gene knockout techniques and shuttle vectors are being developed at an increasing pace. For S. solfataricus knockout systems as well as different shuttle vectors are available. For the genetically more stable S. acidocaldarius shuttle vectors have been recently developed. In this review we summarize the currently available genetic tools and methods for the genus Sulfolobus. Different transformation protocols are discussed, as well as all so far developed knockout systems and Sulfolobus-Escherichia coli shuttle vectors are summarized. Special emphasis is put on the important vector components, i.e., selectable markers and Sulfolobus replicons. Additionally, the information gathered on different Sulfolobus strains with respect to their use as recipient strains is reviewed. The advantages and disadvantages of the different systems are discussed and aims for further improvement of genetic systems are identified.     

Herpesvirus genetics has come of age

The genetic analysis of the large and complex herpesviruses has been a constant challenge to herpesvirologists. Elegant methods have been developed to produce mutants in infected cells that rely on the cellular recombination machinery. Bacterial artificial chromosomes (BACs), single copy F-factor-based plasmid vectors of intermediate insert capacity, have now enabled the cloning of complete herpesvirus genomes. Infectious virus genomes can be shuttled between Escherichia coli and eukaryotic cells. Herpesvirus BAC DNA engineering in E. coli by homologous recombination requires neither restriction sites nor cloning steps and allows the introduction of a wide variety of DNA modifications. Such E. coli-based technology has provided a safe, fast and effective approach to the systematic mining of the information stored in herpesvirus genomes as a result of their intimate co-evolution with their specific hosts for millions of years. Use of this technique could lead to new developments in clinical virology and basic virology research, and increase the usage of viral genomes as investigative tools and vectors.     

A review of food-grade vectors in lactic acid bacteria: from the laboratory to their application

Lactic acid bacteria (LAB) have a long history of use in fermented foods and as probiotics. Genetic manipulation of these microorganisms has great potential for new applications in food safety, as well as in the development of improved food products and in health. While genetic engineering of LAB could have a major positive impact on the food and pharmaceutical industries, progress could be prevented by legal issues related to the controversy surrounding this technology. The safe use of genetically modified LAB requires the development of food-grade cloning systems containing only the DNA from homologous hosts or generally considered as safe organisms, and not dependent antibiotic markers. The rationale for the development of cloning vectors derived from cryptic LAB plasmids is the need for new genetic engineering tools, therefore a vision from cryptic plasmids to applications in food-grade vectors for LAB plasmids is shown in this review. Replicative and integrative vectors for the construction of food-grade vectors, and the relationship between resistance mechanism and expression systems, will be treated in depth in this paper. Finally, we will discuss the limited use of these vectors, and the problems arising from their use.     

Diptera vectors of avian Haemosporidian parasites: untangling parasite life cycles and their taxonomy

Haemosporida is a large group of vector-borne intracellular parasites that infect amphibians, reptiles, birds, and mammals. This group includes the different malaria parasites (Plasmodium spp.) that infect humans around the world. Our knowledge on the full life cycle of these parasites is most complete for those parasites that infect humans and, to some extent, birds. However, our current knowledge on haemosporidian life cycles is characterized by a paucity of information concerning the vector species responsible for their transmission among vertebrates. Moreover, our taxonomic and systematic knowledge of haemosporidians is far from complete, in particular because of insufficient sampling in wild vertebrates and in tropical regions. Detailed experimental studies to identify avian haemosporidian vectors are uncommon, with only a few published during the last 25 years. As such, little knowledge has accumulated on haemosporidian life cycles during the last three decades, hindering progress in ecology, evolution, and systematic studies of these avian parasites. Nonetheless, recently developed molecular tools have facilitated advances in haemosporidian research. DNA can now be extracted from vectors' blood meals and the vertebrate host identified; if the blood meal is infected by haemosporidians, the parasite's genetic lineage can also be identified. While this molecular tool should help to identify putative vector species, detailed experimental studies on vector competence are still needed. Furthermore, molecular tools have helped to refine our knowledge on Haemosporida taxonomy and systematics. Herein we review studies conducted on Diptera vectors transmitting avian haemosporidians from the late 1800s to the present. We also review work on Haemosporida taxonomy and systematics since the first application of molecular techniques and provide recommendations and suggest future research directions. Because human encroachment on natural environments brings human populations into contact with novel parasite sources, we stress that the best way to avoid emergent and reemergent diseases is through a program encompassing ecological restoration, environmental education, and enhanced understanding of the value of ecosystem services.     

Geminivirus-based vectors for gene silencing in Arabidopsis

Gene silencing, or RNA interference, is a powerful tool for elucidating gene function in Caenorhabditis elegans and Drosophila melanogaster. The vast genetic, developmental and sequence information available for Arabidopsis thaliana makes this an attractive organism in which to develop reliable gene-silencing tools for the plant world. We have developed a system based on the bipartite geminivirus cabbage leaf curl virus (CbLCV) that allows silencing of endogenous genes singly or in combinations in Arabidopsis. Two vectors were tested: a gene-replacement vector derived from the A component; and an insertion vector derived from the B component. Extensive silencing was produced in new growth from the A component vectors, while only minimal silencing and symptoms were seen in the B component vector. Two endogenous genes were silenced simultaneously from the A component vector and silencing of the genes was maintained throughout new growth. Because the CbLCV vectors are DNA vectors they can be inoculated directly from plasmid DNA. Introduction of these vectors into intact plants bypasses transformation and extends the kinds of silencing studies that can be carried out in Arabidopsis.     

Development of Dual-Activity Vectors by Co-Envelopment of Adenovirus and SiRNA in Artificial Lipid Bilayers

Gene therapy with human adenovirus type 5 (Ad5) has been extensively explored for the treatment of diseases resistant to traditional therapies. Intravenous administration leads to rapid clearance from blood circulation and high liver accumulation, which restrict the use of Ad-based vectors in clinical gene therapy protocols that involve systemic administration. We have previously proposed that such limitations can be improved by engineering artificial lipid envelopes around Ad and designed a variety of artificial lipid bilayer envelopes around the viral capsid. In this study, we sought to explore further opportunities that the artificially enveloped virus constructs could offer, by designing a previously unreported gene therapy vector by simultaneous envelopment of Ad and siRNA within the same lipid bilayer. Such a dual-activity vector can offer efficacious therapy for different genetic disorders where both turning on and switching off genes would be needed. Dynamic light scattering, transmission electron microscopy and atomic force microscopy were used to characterize these vectors. Agarose gel electrophoresis, Ribo green and dot blot assays showed that siRNA and Ad virions can be enveloped together within lipid bilayers at high envelopment efficiency. Cellular uptake and in vitro transfection experiments were carried out to show the feasibility of combining siRNA-mediated gene silencing with viral gene transfer using these newly designed dual-activity vectors.     

乳酸菌食品级表达载体的研究与应用

乳酸菌是能够发酵糖类产生大量有机酸的革兰氏阳性菌的通称,在发酵食品中有着悠久的应用历史。乳酸菌通常被认为是安全菌株,这些微生物的基因工程操作在食品、医学等方面具有广阔的应用前景。表达载体是基因工程中常用的工具之一,大多数乳酸菌的表达载体通常以抗生素抗性基因作为选择标记,然而抗性基因具有潜在的转移性,因此需要开发食品级表达载体。食品级表达载体不含有抗生素的抗性基因,仅包含来自同源宿主或通常被认为是安全生物的DNA。本文介绍了乳酸菌食品级表达载体的构成及其常用宿主,同时对乳酸菌食品级表达载体的应用进行了归纳总结。     

One step construction of Agrobacterium-Recombination-ready-plasmids (OSCAR), an efficient and robust tool for ATMT based gene deletion construction in fungi

Increasing availability of genomic data and sophistication of analytical methodology in fungi has elevated the need for functional genomics tools in these organisms. Previously we reported a method called DelsGate for rapid preparation of deletion constructs for protoplast-mediated fungal transformation systems, which is based on Gateway? technology. However, over the past several years Agrobacteriumtumefaciens-mediated transformation (ATMT) has become the preferred genetic transformation method for an increasing number of fungi. Therefore, we developed a method for One Step Construction of Agrobacterium-Recombination-ready-plasmids (OSCAR), to rapidly create deletion constructs for ATMT systems. The OSCAR methodology involves PCR amplification of the upstream and downstream flanks of the gene of interest, using gene specific primers each with a 5' extension containing one of four different attB recombination sites, modified from the Invitrogen MultiSite Gateway? system. Amplified gene flanks are then mixed with specifically designed marker and binary vectors and treated with BP clonase, generating the deletion construct in a single cloning step. The entire process of deletion construct preparation can be accomplished in just 2days. Using OSCAR we generated eight targeted deletion constructs and used two of them to generate deletion mutants in Verticillium dahliae by ATMT. In summary, OSCAR methodology combines PCR and Gateway? technology to rapidly and robustly generate precise deletion constructs for fungal ATMT and homologous gene replacement.     

Alternative-splicing-based bicistronic vectors for ratio-controlled protein expression and application to recombinant antibody production

In the last decade polycistronic vectors have become essential tools for both basic science and gene therapy applications. In order to co-express heterologous polypeptides, different systems have been developed from Internal Ribosome Entry Site (IRES) based vectors to the use of the 2A peptide. Unfortunately, these methods are not fully suitable for the efficient and reproducible modulation of the ratio between the proteins of interest. Here we describe a novel bicistronic vector type based on the use of alternative splicing. By modifying the consensus sequence that governs splicing, we demonstrate that the ratio between the synthesized proteins could easily vary from 1 : 10 to 10 : 1. We have established this system with luciferase genes and we extended its application to the production of recombinant monoclonal antibodies. We have shown that these vectors could be used in several typical cell lines with similar efficiencies. We also present an adaptation of these vectors to hybrid alternative splicing/IRES constructs that allow a ratio-controlled expression of proteins of interest in stably transfected cell lines.     

Delivery of multiple transgenes to plant cells by an improved version of MultiRound Gateway technology

At present, only few methods for the effective assembly of multigene constructs have been described. Here we present an improved version of the MultiRound Gateway technology, which facilitates plant multigene transformation. The system consists of two attL-flanked entry vectors, which contain an attR cassette, and a transformation-competent artificial chromosome based destination vector. By alternate use of the two entry vectors, multiple transgenes can be delivered sequentially into the Gateway-compatible destination vector. Multigene constructs that carried up to seven transgenes corresponding to more than 26 kb were assembled by seven rounds of LR recombination. The constructs were successfully transformed into tobacco plants and were stably inherited for at least two generations. Thus, our system represents a powerful, highly efficient tool for multigene plant transformation and may facilitate genetic engineering of agronomic traits or the assembly of genetic pathways for the production of biofuels, industrial or pharmaceutical compounds in plants.