植物CRISPR基因组编辑技术的新进展

在过去的4年中,CRISPR/Cas9基因组编辑技术成为生命科学领域的革命性工具,为植物学基础研究和农作物遗传改良提供了高效、快速而又廉价的遗传操作工具。利用CRISPR/Cas9系统可以实现精准的knock-out和knock-in等遗传操作,也可用于靶向激活或抑制基因的表达。在CRISPR/Cas9被广泛地用于基因组编辑的同时,它的编辑能力、效率和精确度也在不断地改进和完善,特别是CRISPR/Cpf1系统的发掘和单碱基编辑技术的创建,使CRISPR系统正逐步成为一个理想的遗传工程技术平台。此外,利用CRISPR/Cas9技术改良的农作物品种也已经涌现,这必将推动精准基因组编辑技术在农作物遗传改良中的应用和发展。     

A gene network engineering platform for lactic acid bacteria

Recent developments in synthetic biology have positioned lactic acid bacteria (LAB) as a major class of cellular chassis for applications. To achieve the full potential of LAB, one fundamental prerequisite is the capacity for rapid engineering of complex gene networks, such as natural biosynthetic pathways and multicomponent synthetic circuits, into which cellular functions are encoded. Here, we present a synthetic biology platform for rapid construction and optimization of large-scale gene networks in LAB. The platform involves a copy-controlled shuttle for hosting target networks and two associated strategies that enable efficient genetic editing and phenotypic validation. By using a nisin biosynthesis pathway and its variants as examples, we demonstrated multiplex, continuous editing of small DNA parts, such as ribosome-binding sites, as well as efficient manipulation of large building blocks such as genes and operons. To showcase the platform, we applied it to expand the phenotypic diversity of the nisin pathway by quickly generating a library of 63 pathway variants. We further demonstrated its utility by altering the regulatory topology of the nisin pathway for constitutive bacteriocin biosynthesis. This work demonstrates the feasibility of rapid and advanced engineering of gene networks in LAB, fostering their applications in biomedicine and other areas.     

Controlling pathogenic risks of water treatment biotechnologies at the source by genetic editing means

Antimicrobial-resistant pathogens in the environment and wastewater treatment systems, many of which are also important pollutant degraders and are difficult to control by traditional disinfection approaches, have become an unprecedented treat to ecological security and human health. Here, we propose the adoption of genetic editing techniques as a highly targeted, efficient and simple tool to control the risks of environmental pathogens at the source. An ‘all-in-one’ plasmid system was constructed in Aeromonas hydrophila to accurately identify and selectively inactivate multiple key virulence factor genes and antibiotic resistance genes via base editing, enabling significantly suppressed bacterial virulence and resistance without impairing their normal phenotype and pollutant-degradation functions. Its safe application for bioaugmented treatment of synthetic textile wastewater was also demonstrated. This genetic-editing technique may offer a promising solution to control the health risks of environmental microorganisms via targeted gene inactivation, thereby facilitating safer application of water treatment biotechnologies.     

Enhancement of extracellular electron transfer and bioelectricity output by synthetic porin

The microbial fuel cell (MFC), is a promising environmental biotechnology for harvesting electricity energy from organic wastes. However, low bacterial membrane permeability of electron shuttles is a limiting factor that restricts the electron shuttle‐mediated extracellular electron transfer (EET) from bacteria to electrodes, thus the electricity power output of MFCs. To this end, we heterologously expressed a porin protein OprF from Pseudomonas aeruginosa PAO1 into Escherichia coli, which dramatically increased its membrane permeability, delivering a much higher current output in MFCs than its parental strain (BL21). We found that the oprF‐expression strain showed more efficient EET than its parental strain. More strikingly, the enhanced membrane permeability also rendered the oprF‐expression strain an efficient usage of riboflavin as the electron shuttle, whereas its parental strain was incapable of. Our results substantiated that membrane permeability is crucial for the efficient EET, and indicated that the expression of synthetic porins could be an efficient strategy to enhance bioelectricity generation by microorganisms (including electrogenic bacteria) in MFCs. Biotechnol. Bioeng. 2013; 110: 408–416. © 2012 Wiley Periodicals, Inc.     

CRISPR-Cas: an efficient tool for genome engineering of virulent bacteriophages

Bacteriophages are now widely recognized as major players in a wide variety of ecosystems. Novel genes are often identified in newly isolated phages as well as in environmental metavirome studies. Most of these novel viral genes have unknown functions but appear to be coding for small, non-structural proteins. To understand their biological role, very efficient genetic tools are required to modify them, especially in the genome of virulent phages. We first show that specific point mutations and large deletions can be engineered in the genome of the virulent phage 2972 using the Streptococcus thermophilus CRISPR-Cas Type II-A system as a selective pressure to increase recombination efficiencies. Of significance, all the plaques tested contained recombinant phages with the desired mutation. Furthermore, we show that the CRISPR-Cas engineering system can be used to efficiently introduce a functional methyltransferase gene into a virulent phage genome. Finally, synthetic CRISPR bacteriophage insensitive mutants were constructed by cloning a spacer-repeat unit in a low-copy vector illustrating the possibility to target multiple regions of the phage genome. Taken together, this data shows that the CRISPR-Cas system is an efficient and adaptable tool for editing the otherwise intractable genomes of virulent phages and to better understand phage-host interactions.     

CRISPR-Cas系统在植物基因组编辑中的研究进展

基因组定点编辑技术是研究基因功能和生物体改造的重要工具。CRISPR-Cas(Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins)系统是近年来发展的一种新型基因组编辑技术,该技术通过一段向导RNA和配套的核酸酶就可对特定的基因组序列进行定点编辑,具有简单高效、应用广泛的特点,受到了生物学家的广泛关注。本文着重介绍CRISPR-Cas系统在植物中的研究进展,包括CRISPR-Cas9系统在植物中的应用与完善、扩大基因组编辑范围的研究、Cas9切口酶和失活酶的拓展、特异性单碱基突变编辑系统的研究、无外源DNA污染的植物基因编辑技术的发展以及基因组编辑技术在作物育种上的应用等方面。同时也提出了还需解决的问题,并展望了基因组编辑系统在作物育种中的应用前景,为开展这一领域的研究工作提供参考。     

Efficient and precise control of gene expression in Geobacter sulfurreducens through new genetic elements and tools for pollutant conversion

Geobacter species, exhibiting exceptional extracellular electron transfer aptitude, hold great potential for applications in pollution remediation, bioenergy production, and natural elemental cycles. Nonetheless, a scarcity of well-characterized genetic elements and gene expression tools constrains the effective and precise fine-tuning of gene expression in Geobacter species, thereby limiting their applications. Here, we examined a suite of genetic elements and developed a new genetic editing tool in Geobacter sulfurreducens to enhance their pollutant conversion capacity. First, the performances of the widely used inducible promoters, constitutive promoters, and ribosomal binding sites (RBSs) elements in G. sulfurreducens were quantitatively evaluated. Also, six native promoters with superior expression levels than constitutive promoters were identified on the genome of G. sulfurreducens. Employing the characterized genetic elements, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system was constructed in G. sulfurreducens to achieve the repression of an essential gene-aroK and morphogenic genes-ftsZ and mreB. Finally, applying the engineered strain to the reduction of tungsten trioxide (WO₃), methyl orange (MO), and Cr(VI), We found that morphological elongation through ftsZ repression amplified the extracellular electron transfer proficiency of G. sulfurreducens and facilitated its contaminant transformation efficiency. These new systems provide rapid, versatile, and scalable tools poised to expedite advancements in Geobacter genomic engineering to favor environmental and other biotechnological applications.     

生物工程强化微生物电合成转化CO_2的研究进展

微生物电合成(Microbial electrosynthesis,MES)可直接利用电能驱动微生物还原固定CO_2合成多碳化合物,为可再生新能源转化、精细化学品制备和生态环境保护提供新机遇。但是,微生物吸收胞外电极电子速率慢、产物合成效率低和产品品位不高,限制了MES实现工业化应用。在概述阴极电活性微生物吸收胞外电子的分子机制的基础上,重点综述近5年应用生物工程的理论和技术强化MES用于CO_2转化的策略与研究进展,包括改造和调控胞外电子传递通路和胞内代谢途径以及定向构建有限微生物混合培养菌群三方面,阐明了生物工程可有效突破MES中电子传递慢和可用代谢途径相对单一等瓶颈。针对目前生物工程在改进MES所面临的主要问题,从胞外电子传递机理研究、基因工具箱开发、组学技术与现代分析技术联用等角度展望了今后的研究方向。     

Microbial extracellular electron transfer and its relevance to iron corrosion

Extracellular electron transfer (EET) is a microbial metabolism that enables efficient electron transfer between microbial cells and extracellular solid materials. Microorganisms harbouring EET abilities have received considerable attention for their various biotechnological applications, including bioleaching and bioelectrochemical systems. On the other hand, recent research revealed that microbial EET potentially induces corrosion of iron structures. It has been well known that corrosion of iron occurring under anoxic conditions is mostly caused by microbial activities, which is termed as microbiologically influenced corrosion (MIC). Among diverse MIC mechanisms, microbial EET activity that enhances corrosion via direct uptake of electrons from metallic iron, specifically termed as electrical MIC (EMIC), has been regarded as one of the major causative factors. The EMIC‐inducing microorganisms initially identified were certain sulfate‐reducing bacteria and methanogenic archaea isolated from marine environments. Subsequently, abilities to induce EMIC were also demonstrated in diverse anaerobic microorganisms in freshwater environments and oil fields, including acetogenic bacteria and nitrate‐reducing bacteria. Abilities of EET and EMIC are now regarded as microbial traits more widespread among diverse microbial clades than was thought previously. In this review, basic understandings of microbial EET and recent progresses in the EMIC research are introduced.     

微生物胞外电子传递效率的合成生物学强化

电活性微生物(产电微生物和亲电微生物)通过与外界环境进行双向电子和能量传递来实现多种微生物电催化过程(包括微生物燃料电池、微生物电解电池、微生物电催化等),从而实现在环境、能源领域的广泛应用,并为开发有效且可持续性生产新能源或大宗精细化学品的工艺提供了新机会。但是,电活性微生物的胞外电子传递效率比较低,这已经成为限制微生物电催化系统在工业应用中的主要瓶颈。以下综述了近年来利用合成生物学改造电活性微生物的相关研究成果,阐明了合成生物学如何用于打破电活性微生物胞外电子传递途径低效率的瓶颈,从而实现电活性微生物与环境的高效电子传递和能量交换,推动电活性微生物电催化系统的实用化进程。     

Genome editing in Bombyx mori: New opportunities for silkworm functional genomics and the sericulture industry

In recent years, research in life sciences has been remarkably revolutionized owing to the establishment, development and application of genome editing technologies. Genome editing has not only accelerated fundamental research but has also shown promising applications in agricultural breeding and therapy. In particular, the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology has become an indispensable tool in molecular biology owing to its high efficacy and simplicity. Genome editing tools have also been established in silkworm (Bombyx mori), a model organism of Lepidoptera insects with high economic importance. This has remarkably improved the level and scope of silkworm research and could reveal new mechanisms or targets in basic entomology and pest management studies. In this review, we summarize the progress and potential of genome editing in silkworm and its applications in functional genomic studies for generating novel genetic materials.     

Efficient Cas9 multiplex editing using unspaced sgRNA arrays engineering in a Potato virus X vector

Systems based on the clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR-associated proteins (Cas) have revolutionized genome editing in many organisms, including plants. Most CRISPR-Cas strategies in plants rely on genetic transformation using Agrobacterium tumefaciens to supply the gene editing reagents, such as Cas nucleases or the synthetic guide RNA (sgRNA). While Cas nucleases are constant elements in editing approaches, sgRNAs are target-specific and a screening process is usually required to identify those most effective. Plant virus-derived vectors are an alternative for the fast and efficient delivery of sgRNAs into adult plants, due to the virus capacity for genome amplification and systemic movement, a strategy known as virus-induced genome editing. We engineered Potato virus X (PVX) to build a vector that easily expresses multiple sgRNAs in adult solanaceous plants. Using the PVX-based vector, Nicotiana benthamiana genes were efficiently targeted, producing nearly 80% indels in a transformed line that constitutively expresses Streptococcus pyogenes Cas9. Interestingly, results showed that the PVX vector allows expression of arrays of unspaced sgRNAs, achieving highly efficient multiplex editing in a few days in adult plant tissues. Moreover, virus-free edited progeny can be obtained from plants regenerated from infected tissues or infected plant seeds, which exhibit a high rate of heritable biallelic mutations. In conclusion, this new PVX vector allows easy, fast and efficient expression of sgRNA arrays for multiplex CRISPR-Cas genome editing and will be a useful tool for functional gene analysis and precision breeding across diverse plant species, particularly in Solanaceae crops.     

非常规酵母的分子遗传学及合成生物学研究进展

先进的合成生物学技术与传统的分子遗传学技术的结合更有助于实现酵母底盘细胞的快速改造和优化。酵母合成生物学研究最早开始于常规酵母——酿酒酵母(Saccharomyces cerevisiae),近些年来又迅速扩展至一些非常规酵母,包括巴斯德毕赤酵母(Pichiapastoris)、解脂耶氏酵母(Yarrowialipolytica)、乳酸克鲁维酵母(Kluyveromyces lactis)和多形汉逊酵母(Hansenula polymorpha)等。借助合成生物学技术与工具,目前科学家们已经成功开发出了能够高效生产生物材料、生物燃料、生物基化学品、蛋白质制剂、食品添加剂和药物等工业产品的重组非常规酵母工程菌株。本文系统总结了合成生物学工具(主要是基因组编辑工具)、合成生物学组件(主要是启动子和终止子)和相关分子遗传学方法在上述非常规酵母系统(底盘细胞)中的最新研究进展和应用情况,并讨论了其他合成生物学技术在这些非常规酵母表达系统中的潜在适用性和应用前景。这为研究人员利用合成生物学方法在这一新型非模式微生物底盘细胞中设计和构建各种高附加值工业产品的异源合成模块并最终实现目标化合物的高效生物合成提供了科学的理论指导。     

Gene Designer: a synthetic biology tool for constructing artificial DNA segments

Background  

Direct synthesis of genes is rapidly becoming the most efficient way to make functional genetic constructs and enables applications such as codon optimization, RNAi resistant genes and protein engineering. Here we introduce a software tool that drastically facilitates the design of synthetic genes.     

CRISPR/Cas基因组编辑技术在乳酸菌中的应用及研究展望

乳酸菌是一类重要的食品工业微生物,目前对其功能基因鉴定和挖掘优良功能基因主要依赖于传统的基因同源重组技术,该技术尽管有较高的可靠性,但是存在操作繁琐、效率低下等不足,严重制约了乳酸菌优良菌株的遗传选育。CRISPR/Cas基因编辑技术极大提升了对多物种基因组的编辑效率,这为乳酸菌功能基因的快速鉴定及遗传改良提供了可能,但是现有的CRISPR/Cas基因编辑技术在乳酸菌的应用还存在诸多限制。本文综述了CRISPR/Cas基因编辑技术在乳酸菌基因组上的应用现状及亟待解决的问题,并展望了乳酸菌基因组编辑技术的未来发展趋势,为乳酸菌功能基因鉴定及遗传改良提供参考。     

后基因组时代基因组重排在微生物菌种选育中的应用及展望

基因组重排作为一种实用高效的育种技术,在缺乏遗传背景认知和可操作遗传体系等条件下,可以突破微生物种属间的限制,经过多轮递推的原生质体融合来加速其人工定向进化,在微生物菌种改良及代谢产物开发和产业化等研究领域得到了广泛应用。步入后基因组时代,快速发展的组学和生物信息学使基因组重排成为连接各种微生物育种方法的重要纽带,为我们深入探索微生物复杂的代谢网络和全局调控机制,更为精准地实施对微生物的人工调控和定向进化提供了契机。本文系统性地回顾了近年来基因组重排在微生物菌种选育中的应用研究,尤其针对围绕其开展的组学研究进行了详细阐述,并对基因组重排与组学、生物信息学和合成生物学等新兴技术的联合应用进行了展望。     

基因组工程在医学合成生物学中的应用

合成生物学旨在建立一套完整的工程理论和方法,通过设计和组装基本生物学元件,更为有效地实现复杂生物系统的设计,并使其完成可编程的生物学功能。近年来随着可编程基因组元件的出现,特别是CRISPR和CRISPRi技术平台的建立和完善,使得合成生物学进入了一个全新发展的时期。本文重点综述CRISPR等基因组编辑和调控技术,其在构建可编程生物学元件和复杂基因线路的应用以及合成生物学在医学中(称为医学合成生物学)的发展前景。     

CRISPR/Cas基因编辑系统在原核微生物细胞工厂构建中的开发与应用

随着能源和环境问题的日益突出,化学品以及燃料的合成方式正逐渐由传统的化学法合成转变为以细菌为基础的生物炼制过程,其中最关键问题是需要开发出合适的基因工程工具用于构建相应的产品生产菌株。成簇的规律间隔短回文重复序列(Clusteredregularlyinterspacedshortpalindromic repeats,CRISPR)/CRISPR相关蛋白(CRISPR-associated proteins,Cas)系统是一种存在于细菌和古细菌中的免疫系统,能够用于抵御病毒和外源质粒的入侵,近年来被开发成为一种高效、便捷、精确的基因编辑工具,显示出巨大的应用潜力。本文立足于CRISPR/Cas系统的原理与最新分类,结合实例综述了CRISPR/Cas基因编辑系统在原核微生物细胞工厂构建中的建立与优化策略,以及主要的应用方向,并探讨该系统所面临的主要问题并提出了一些可行的解决方案。