基于双碱基编辑系统的植物基因靶向随机突变技术

遗传性变异是表型多样性的基础,靶向饱和突变作物基因可以促进产生具有优异农艺性状的突变体。相较于传统诱变育种和异源物种中的定向进化方法,基于双碱基编辑系统的植物基因靶向随机突变技术可对植物内源基因产生高效突变,从而实现原位定向进化,加快植物育种及功能基因研究进程。该文介绍了使用饱和靶向内源基因突变编辑器(STEME)对植...     

单碱基编辑系统介导欧拉藏绵羊GDF9、FecB多胎基因的定点突变

本研究旨在利用单碱基编辑系统(single base editing system)实现欧拉藏绵羊成纤维细胞FecB和GDF9基因靶位点A到G和C到T的碱基替换并检测其编辑效率。首先设计合成靶向欧拉藏绵羊FecB和GDF9基因的sgRNA序列,再分别连接至epi-ABEmax、epi-BE4max质粒,构建载体并电转至欧拉藏绵羊成纤维细胞,最后对阳性细胞FecB和GDF9基因进行Sanger测序鉴定靶位点突变结果,并通过T-A克隆估算单碱基编辑系统的编辑效率。结果显示获得了靶向欧拉藏绵羊FecB和GDF9基因的sgRNA,并构建使欧拉藏绵羊FecB和GDF9基因单碱基突变的载体,FecB基因靶位点编辑效率为39.13%,GDF9基因靶位点(G260、G721、G1184)编辑效率分别为10.52%、26.67%和8.00%。本研究运用单碱基编辑系统在欧拉藏绵羊成纤维细胞上实现了FecB和GDF9基因靶位点突变,为改良欧拉藏绵羊一胎多羔的繁殖性状奠定理论基础。     

碱基编辑技术的发展与应用

碱基编辑技术,以CRISPR/Cas系统为平台,引导胞嘧啶脱氨酶或腺嘌呤脱氨酶至特定的基因组靶点,产生靶向性的C至T或者A至G的碱基转换。自碱基编辑技术问世以来,全球多个科研团队通过优化改进得到了一系列高精准性、广靶向性、小编辑框、普适性的碱基编辑器。在应用方面,碱基编辑器能够在人体细胞、动植物细胞以及胚胎中进行高效的碱基转换,在治疗人类遗传病、构建动物疾病模型、植物育种等方面具有巨大的应用潜能。本文就碱基编辑技术的发展、优化和应用等方面进行综述和展望。     

人类胚胎单碱基编辑治疗遗传疾病的研究

基因编辑技术是当今生物学研究领域最为重要的颠覆性技术之一,以CRISPR/Cas9系统为核心的基因编辑工具被广泛应用于包括人类体细胞、生殖细胞编辑相关的医学研究领域。虽然CRISPR/Cas9系统可以高效编辑靶基因,但其精准编辑能力依赖于效率极低的同源重组方式,这极大限制了其定点编辑的能力与应用范围,所以寻找一种能高效引入点突变的新型基因编辑工具具有重大的应用价值。以CRISPR/Cas9系统为基础的单碱基编辑系统可以在基因组靶位点实现精准、高效的C/G和T/A碱基间的转换,其编辑能力已经在动植物、人体细胞以及人类胚胎中得到证实。利用单碱基编辑技术,有望对人类超过70%的相关遗传性致病位点进行修复。现就人类胚胎单碱基编辑治疗遗传疾病的最新研究进展进行综述和展望。     

AID介导的原位靶点突变——哺乳动物DNA碱基编辑新技术

单核苷酸的多样性是遗传多样性的主要来源,是人类个体差异的重要遗传学基础,同时也是分子进化的动力和很多疾病的直接诱因。然而,在哺乳动物中,仍然缺乏有效诱导单核苷酸的突变的工具,无法通过实验高效和高通量地研究单核苷酸突变的功能。现有的大部分实验技术只能扰乱基因的功能或者表达,造成基因功能缺失,对诱导新功能的获得无能为力。而利用靶向性胞嘧啶脱氨酶介导的碱基编辑(targeted AID-mediated mutagenesis,TAM)技术,可以在sg RNA靶向的基因组DNA上,将胞嘧啶和鸟嘌呤随机地向其他三个碱基转变,因而产生海量的突变体,结合遗传筛选,从而分析单核苷酸突变的功能或诱导蛋白质的体内进化。同时,在一种多肽抑制剂(uracil glycosylase inhibitor,UGI)的辅助下,TAM可以诱导特定的胞嘧啶向胸腺嘧啶转变,实现单碱基的精确编辑,为治疗单核苷酸突变诱导的遗传病提供方案。利用这项技术,已经在慢性骨髓瘤细胞中,成功筛选出已报道的以及新的Imatinib耐药性位点。因此,作为高效的哺乳动物DNA碱基编辑新技术,TAM可以广泛应用于蛋白质工程、分子遗传学研究和基因治疗等领域。     

主编导读

<正>本期主编导读主题：重要农艺性状功能基因及植物-微生物互作机制、病毒检测及疫苗研究、微生物与环境、多方位及混合式教学模式。重要农艺性状功能基因及植物-微生物互作机制阐明作物重要农艺性状分子控制的机理,以及植物-微生物(包括有益微生物和病原微生物)互作机制,能够为分子改良和设计重要农艺性状奠定理论基础,进而推动我国育种科学的可持续发展。     

BE3型胞嘧啶碱基编辑器在谷氨酸棒杆菌中的开发及应用

碱基编辑技术结合了CRISPR/Cas系统的靶向特异性与碱基脱氨酶的催化活性,因其不产生双链DNA断裂、不需要外源DNA模板、不依赖同源重组修复,自开发以来,便受到研究者的追捧,在哺乳动物细胞、植物、微生物等领域相继得到开发与应用。为了进一步丰富碱基编辑系统在谷氨酸棒杆菌中的应用,将鼠源胞嘧啶脱氨酶(rAPOBEC1)与nCas9蛋白融合,实现了在谷氨酸棒杆菌中C到T的编辑,编辑比例较低(0-20%);在上述融合蛋白C端添加UGI蛋白,构建BE3型胞嘧啶碱基编辑器,抑制体内的DNA碱基切除修复机制,显著的提高了碱基编辑效率,使得C到T的碱基编辑效率高达90%;为了简化操作,将双质粒碱基编辑系统优化为单质粒碱基编辑系统,并显著提高转化效率;最后通过单质粒碱基编辑系统对基因组中其他位点的编辑测试,进一步证明了BE3型碱基编辑器在谷氨酸棒杆菌中的高效性,同时发现该碱基编辑器具有较宽的编辑窗口(PAM上游-11到-19位),有助于覆盖更多的基因组靶标位点,为谷氨酸棒杆菌的基因组改造提供了更多的工具选择。     

CRISPR/Cas9编辑棉花精氨酸酶基因促进侧根形成和发育

基因敲除是植物功能基因组研究和农艺性状定向遗传改良的关键步骤,近年来,CRISPR/Cas9介导的目标基因敲除在拟南芥(Arabidopsis thaliana)、水稻(Oryza sativa)等植物中取得飞速的发展,然而在棉花(Gossypium spp.)中目前尚没有成功的报道.陆地棉(G.hirsutum L.)是典型的异源四倍体模式作物,包括A亚基因组和D亚基因组.利用CRISPR/Cas9技术同时成功地敲除A和D亚基因组精氨酸酶编码基因(GhARG),Gh_A05G2143和Gh_D05G2397.无论高氮还是低氮条件下,T1代双基因纯和株系侧根生长发育显著地提高.因此,本研究的CRISPR/Cas9系统可以同时高效地编辑棉花中多个同源基因,从而实现关键农艺性状简单快速得改良.     

基因编辑之“新宠”—单碱基基因组编辑系统

近年发展起来的人工核酸酶可通过引起特定位点的DNA双链断裂实现对目的片段的有效编辑。为进一步提高碱基修改的效率和精确度,2016年研究者们利用CRISPR/Cas9识别特定DNA序列的功能,结合胞嘧啶脱氨酶的生化活性发明了将胞嘧啶高效转换为胸腺嘧啶(C>T)的嘧啶单碱基编辑系统(base editor)。这一系统虽然能精准实现嘧啶直接转换,大大提高精确基因编辑效率,但美中不足的是无法对嘌呤进行修改。近期,Nature报道了将细菌中的tRNA腺嘌呤脱氨酶定向进化形成具有催化DNA腺嘌呤底物的脱氨酶,将其与Cas9系统融合发明了具有高效催化腺嘌呤转换为鸟嘌呤的新工具—腺嘌呤单碱基编辑系统(ABEs, adenine base editors)。本文总结了单碱基编辑工具的发展历程和最新研究进展,着重介绍ABEs的研发过程,并对单碱基编辑工具今后的应用方向和研发方向进行展望。     

Efficient genome editing and gene knockout in Setaria viridis with CRISPR/Cas9 directed gene editing by the non-homologous end-joining pathway

The CRISPR/Cas9 system has been used for genome editing in several organisms, including higher plants. This system induces site-specific mutations in the genome based on the nucleotide sequence of engineered guide RNAs. The complex genomes of C4 grasses makes genome editing a challenge in key grass crops like maize (Zea mays), sorghum (Sorghum bicolor), Brachiaria spp., switchgrass (Panicum virgatum), and sugarcane (Saccharum spp.). Setaria viridis is a diploid C4 grass widely used as a model for these C4 crop plants. Here, an optimized CRISPR/Cas9 binary vector that exploits the non-homologous end joining (NHEJ) system was used to knockout a green fluorescent protein (gfp) transgene in S. viridis accession A10.1. Transformation of embryogenic callus by A. tumefaciens generated ten glufosinate-ammonium resistant transgenic events. In the T0 generation, 60% of the events were biallelic mutants in the gfp transgene with no detectable accumulation of GFP protein and without insertions or deletions in predicted off-target sites. The gfp mutations generated by CRISPR/Cas9 were stable and displayed Mendelian segregation in the T1 generation. Altogether, the system described here is a highly efficient genome editing system for S. viridis, an important model plant for functional genomics studies in C4 grasses. Also, this system is a potential tool for improvement of agronomic traits in C4 crop plants with complex genomes.     

CRISPR-Cas系统在植物中的研究进展与监管政策

基因编辑技术作为一种颠覆性新技术,现已广泛应用于作物的遗传改良,显示出巨大的发展潜力和应用价值。各国在加快技术研发的同时也十分关注其可能带来的安全性问题,相继出台了基因编辑作物的安全监管政策。综述了目前常用的CRISPR基因编辑系统的原理, 最新开发的一系列CRISPR变体,CRISPR系统在植物中的应用,基因编辑植物检测方法及国际上的相关监管政策,以期为我国基因编辑作物监管政策的制定提供理论数据。     

The evolution of chloroplast RNA editing

RNA editing alters the nucleotide sequence of an RNA molecule so that it deviates from the sequence of its DNA template. Different RNA-editing systems are found in the major eukaryotic lineages, and these systems are thought to have evolved independently. In this study, we provide a detailed analysis of data on C-to-U editing sites in land plant chloroplasts and propose a model for the evolution of RNA editing in land plants. First, our data suggest that the limited RNA-editing system of seed plants and the much more extensive systems found in hornworts and ferns are of monophyletic origin. Further, although some eukaryotic editing systems appear to have evolved to regulate gene expression, or at least are now involved in gene regulation, there is no evidence that RNA editing plays a role in gene regulation in land plant chloroplasts. Instead, our results suggest that land plant chloroplast C-to-U RNA editing originated as a mechanism to generate variation at the RNA level, which could complement variation at the DNA level. Under this model, many of the original sites, particularly in seed plants, have been subsequently lost due to mutation at the DNA level, and the function of extant sites is merely to conserve certain codons. This is the first comprehensive model for the evolution of the chloroplast RNA-editing system of land plants and may also be applicable to the evolution of RNA editing in plant mitochondria.     

RNA编辑的研究进展

RNA编辑,即通过碱基的插入、删除和替换对RNA进行的转录后加工过程,这一表观遗传现象也被认为是在RNA水平上对遗传信息进行修复的一种修正机制.本文主要综述了目前植物中基于PPR基因家族等编辑复合体以及动物中关于CRISPR/Cas系统的两种RNA编辑系统,并介绍了RNA编辑在植物生长发育过程中的重要作用,并展望了RN...     

Oligonucleotide‐directed mutagenesis for precision gene editing

Differences in gene sequences, many of which are single nucleotide polymorphisms, underlie some of the most important traits in plants. With humanity facing significant challenges to increase global agricultural productivity, there is an urgent need to accelerate the development of these traits in plants. oligonucleotide‐directed mutagenesis (ODM), one of the many tools of Cibus’ Rapid Trait Development System ( RTDS ^?) technology, offers a rapid, precise and non‐transgenic breeding alternative for trait improvement in agriculture to address this urgent need. This review explores the application of ODM as a precision genome editing technology, with emphasis on using oligonucleotides to make targeted edits in plasmid, episomal and chromosomal DNA of bacterial, fungal, mammalian and plant systems. The process of employing ODM by way of RTDS technology has been improved in many ways by utilizing a fluorescence conversion system wherein a blue fluorescent protein (BFP) can be changed to a green fluorescent protein (GFP) by editing a single nucleotide of the BFP gene (CAC→TAC; H66 to Y66). For example, dependent on oligonucleotide length, applying oligonucleotide‐mediated technology to target the BFP transgene in Arabidopsis thaliana protoplasts resulted in up to 0.05% precisely edited GFP loci. Here, the development of traits in commercially relevant plant varieties to improve crop performance by genome editing technologies such as ODM, and by extension RTDS , is reviewed.     

Efficient Library Construction by In Vivo Recombination with a Telomere-Originated Autonomously Replicating Sequence of Hansenula polymorpha

A high frequency of transformation and an equal gene dosage between transformants are generally required for activity-based selection of mutants from a library obtained by directed evolution. An efficient library construction method was developed by using in vivo recombination in Hansenula polymorpha. Various linear sets of vectors and insert fragments were transformed and analyzed to optimize the in vivo recombination system. A telomere-originated autonomously replicating sequence (ARS) of H. polymorpha, reported as a recombination hot spot, facilitates in vivo recombination between the linear transforming DNA and chromosomes. In vivo recombination of two linear DNA fragments containing the telomeric ARS drastically increases the transforming frequency, up to 10-fold, compared to the frequency of circular plasmids. Direct integration of the one-end-recombined linear fragment into chromosomes produced transformants with single-copy gene integration, resulting in the same expression level for the reporter protein between transformants. This newly developed in vivo recombination system of H. polymorpha provides a suitable library for activity-based selection of mutants after directed evolution.     

Efficient library construction by in vivo recombination with a telomere-originated autonomously replicating sequence of Hansenula polymorpha

A high frequency of transformation and an equal gene dosage between transformants are generally required for activity-based selection of mutants from a library obtained by directed evolution. An efficient library construction method was developed by using in vivo recombination in Hansenula polymorpha. Various linear sets of vectors and insert fragments were transformed and analyzed to optimize the in vivo recombination system. A telomere-originated autonomously replicating sequence (ARS) of H. polymorpha, reported as a recombination hot spot, facilitates in vivo recombination between the linear transforming DNA and chromosomes. In vivo recombination of two linear DNA fragments containing the telomeric ARS drastically increases the transforming frequency, up to 10-fold, compared to the frequency of circular plasmids. Direct integration of the one-end-recombined linear fragment into chromosomes produced transformants with single-copy gene integration, resulting in the same expression level for the reporter protein between transformants. This newly developed in vivo recombination system of H. polymorpha provides a suitable library for activity-based selection of mutants after directed evolution.     

Canadian regulatory aspects of gene editing technologies

The development of gene editing techniques, capable of producing plants and animals with new and improved traits, is revolutionizing the world of plant and animal breeding and rapidly advancing to commercial reality. However, from a regulatory standpoint the Government of Canada views gene editing as another tool that will join current methods used to develop desirable traits in plants and animals. This is because Canada focusses on the potential risk resulting from the novelty of the trait, or plant or animal product entering the Canadian environment or market place, rather than the process or method by which it was created. The Canadian Food Inspection Agency is responsible for the regulation of the environmental release of plants with novel traits, and novel livestock feeds, while Health Canada is responsible for the regulation of novel foods. Environment and Climate Change Canada, in partnership with Health Canada, regulates modified animals for entry into the environment. In all cases, these novel products may be the result of conventional breeding, mutagenesis, recombinant DNA techniques or other methods of plant or animal breeding such as gene editing. This novelty approach allows the Canadian regulatory system to efficiently adjust to any new developments in the science of plant and animal breeding and allows for risk-appropriate regulatory decisions. This approach encourages innovation while maintaining science-based regulatory expertise. Canadian regulators work cooperatively with proponents to determine if their gene editing-derived product meets the definition of a novel product, and whether it would be subject to a pre-market assessment. Therefore, Canada’s existing regulatory system is well positioned to accommodate any new innovations or technologies in plant or animal breeding, including gene editing.

     

谷氨酸棒杆菌基因编辑的研究进展

谷氨酸棒杆菌是生产氨基酸、有机酸等的重要菌株,广泛应用于食品、医药领域。利用基因编辑技术对谷氨酸棒杆菌进行基因功能研究,在提高目的产物产量、发现新的基因功能等方面有重要意义。近年来,基因编辑技术发展日新月异,从基于同源重组的传统基因编辑技术到以人工核酸酶介导的基因编辑均在谷氨酸棒杆菌中得到合理应用。其中,CRISPR技术以其快速、简便、编辑效率高等优点成为现阶段研究者用于改造谷氨酸棒杆菌的主要技术,但是更为简单、高效的编辑手段依旧需要进一步研究开发,以获得优良菌株应用于工业生产中。