STEME系统:一种助力体内定向进化的新工具

通过定向进化(directed evolution)可以快速进行蛋白工程改良及重要基因功能研究,以获得新型农艺性状突变体。近期,中国科学院遗传与发育生物学研究所高彩霞团队和李家洋团队合作构建了新型的饱和靶向内源诱变编辑器(saturated targeted endogenous mutagenesis editors, STEMEs),并在植物中实现了基因的定向进化和功能筛选。该系统融合了现有的2种单碱基编辑技术,成功实现在植物体内同时诱导C:G>T:A、A:T>G:C双碱基编辑,通过靶向OsACC羧基转移酶结构域编码序列定向进化出水稻除草剂抗性植株。这种在体内进行基因定向进化的新方法,对于今后农作物重要农艺性状的筛选和功能基因研究具有重要作用。本文对STEME系统的组成、编辑效率和应用原理进行介绍,并与已有的定向进化方法进行比较,为加速作物种质资源创新研究提供参考。     

锌指核酸内切酶:基因操作的有力工具

针对动植物进行基因靶向操作的技术,是解析基因功能、研究疾病,以及农业经济生产中一个有用的工具。至今,基因靶向操作主要是通过在胚胎干细胞(embryonic stem cell,ES cell)中进行同源重组或者是体细胞核转移的方法进行,但同源重组方法由于需要ES细胞而被限制在个别物种,而核转移方法存在核去分化、效率低、成本高的缺陷。近几年,一种基于锌指核酸内切酶(zinc-finger nuclease,ZFN)基因靶向修饰的新技术被应用于包括植物、果蝇、爪蟾、斑马鱼和大鼠等不同物种的基因操作。通过胚胎注射ZFN的质粒或是mRNA可以有效地定靶并迅速地在内源基因上引起可遗传的突变。ZFN介导基因靶向敲除的可行性,使得那些无法获得ES细胞和克隆技术支持的物种的基因靶向修饰成为可能。     

蛋白质体外定向进化策略研究进展

定向进化已广泛应用于蛋白质的分子改造,是改善蛋白质特性的最有效策略之一。为了获得更高的突变效率,构建含有更多突变体的文库,不断地发明创新各种高效的蛋白质体外定向进化方法。对近年来出现的几种定向进化方法三核苷酸突变(TriNex)、序列饱和突变(SeSaM)、随机链交换突变法(RAISE)、重叠延伸蛋白域文库法(PDLGO)和迭代饱和突变法(ISM)的原理、特点及应用进行综述。     

人工miRNA技术及其在植物抗病育种中的应用

内源miRNAs被认为是真核生物调控基因表达的重要调节因子,人工miRNA(amiRNA)是一种基于天然miRNA的结构用以沉默内源基因的有效策略。利用人工miRNA替换植物内源miRNA前体中miRNA/miRNA*序列,使产生的人工miRNA与靶基因配对,并特异性地抑制靶基因的表达。从天然miRNA的合成过程和作用机制,人工miRNA的设计基本原理及构建方法,人工miRNA技术在植物抗病育种上的应用等方面进行综述。     

定向进化技术的最新进展

筛选是制约酶定向进化改造的瓶颈。为解决这一难题,近年来一系列基于组合活性中心饱和突变(Combinatorial active-site saturation test,CAST)及迭代饱和突变(Iterative saturation mutagenesis,ISM)的半理性设计新方法被开发出来,包括单密码子饱和突变(Single code saturation mutagenesis,SCSM)、双密码子饱和突变(Double code saturation mutagenesis,DCSM)和三密码子饱和突变(Triple code saturation mutagenesis,TCSM)。通过构建"小而精"的高质量突变体文库,对特定靶点进行组合突变,并成功应用于多种生物催化剂的立体/区域选择性及催化活力等多参数的改造。文中综述了近年来定向进化技术的最新进展及其在生物催化剂定向改造中的应用。     

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。     

用培养细胞筛选对农业有价值的几种突变体

从高等植物细胞培养筛选突变体与体细胞无性系变异、原生质体融合和把外源基因导入植物体,都是在细胞中产生新遗传组合的途径,同属于植物细胞遗传操作的范畴;与育种关系密切。突变育种广泛地包含诱导出突变的任何过程,故Reisch也把体细胞无性系变异视为突变育种的一种形式。十多年来,有些人积极研究用培养细胞选择高等植物突     

CRISPR/Cas基因编辑技术及其在作物育种中的应用

CRISPR/Cas基因编辑技术在植物基因功能研究和作物遗传改良方面具有重要应用价值,其主要依赖gRNA引导核酸内切酶在目标基因组位置产生双链断裂（DSBs）,DSBs在通过非同源末端连接（NHEJ）或同源重组（HDR）方式进行修复时,会引起靶标位置核苷酸序列的缺失、插入或者替换,从而实现基因编辑。介绍了CRISPR/Cas基因编辑技术的作用机理及发展趋势,并对CRISPR/Cas技术在主要粮食及经济作物育种中的应用进展进行了总结,以期为农作物育种提供有益的参考。     

安全标记基因在转基因植物中的应用

转基因植物的抗性标记一直是转基因生物安全性争论的焦点,是限制转基因植物应用的瓶颈之一。筛选安全标记基因替代抗生素标记基因已成为解决转基因植物安全性和促进转基因植物应用的重要策略。综述了生物安全标记基因的产生背景、系统分类、筛选原理及不同起源的标记基因在植物基因工程中的应用和存在问题。选用植物内源标记基因已成为转基因植物安全标记基因研究的重要方向。     

Expanding the base editing scope in rice by using Cas9 variants

Base editing is a novel genome editing strategy that enables irreversible base conversion at target loci without the need for double stranded break induction or homology‐directed repair. Here, we developed new adenine and cytosine base editors with engineered SpCas9 and SaCas9 variants that substantially expand the targetable sites in the rice genome. These new base editors can edit endogenous genes in the rice genome with various efficiencies. Moreover, we show that adenine and cytosine base editing can be simultaneously executed in rice. The new base editors described here will be useful in rice functional genomics research and will advance precision molecular breeding in crops.     

PCRless library mutagenesis via oligonucleotide recombination in yeast

The directed evolution of biomolecules with new functions is largely performed in vitro, with PCR mutagenesis followed by high-throughput assays for desired activities. As synthetic biology creates impetus for generating biomolecules that function in living cells, new technologies are needed for performing mutagenesis and selection for directed evolution in vivo. Homologous recombination, routinely exploited for targeted gene alteration, is an attractive tool for in vivo library mutagenesis, yet surprisingly is not routinely used for this purpose. Here, we report the design and characterization of a yeast-based system for library mutagenesis of protein loops via oligonucleotide recombination. In this system, a linear vector is co-transformed with single-stranded mutagenic oligonucleotides. Using repair of nonsense codons engineered in three different active-site loops in the selectable marker TRP1 as a model system, we first optimized the recombination efficiency. Single-loop recombination was highly efficient, averaging 5%, or 4.0×10(5) recombinants. Multiple loops could be simultaneously mutagenized, although the efficiencies dropped to 0.2%, or 6.0×10(3) recombinants, for two loops and 0.01% efficiency, or 1.5×10(2) recombinants, for three loops. Finally, the utility of this system for directed evolution was tested explicitly by selecting functional variants from a mock library of 1:10(6) wild-type:nonsense codons. Sequencing showed that oligonucleotide recombination readily covered this large library, mutating not only the target codon but also encoded silent mutations on either side of the library cassette. Together these results establish oligonucleotide recombination as a simple and powerful library mutagenesis technique and advance efforts to engineer the cell for fully in vivo directed evolution.     

Directed evolution of transketolase substrate specificity towards an aliphatic aldehyde

Mutants of transketolase (TK) with improved substrate specificity towards the non-natural aliphatic aldehyde substrate propionaldehyde have been obtained by directed evolution. We used the same active-site targeted saturation mutagenesis libraries from which we previously identified mutants with improved activity towards glycolaldehyde, which is C2-hydroxylated like all natural TK substrates. Comparison of the new mutants to those obtained previously reveals distinctly different subsets of enzyme active-site mutations with either improved overall enzyme activity, or improved specificity towards either the C2-hydroxylated or non-natural aliphatic aldehyde substrate. While mutation of phylogenetically variant residues was found previously to yield improved enzyme activity on glycolaldehyde, we show here that these mutants in fact gave improved activity on both substrate types. In comparison, the new mutants were obtained at conserved residues which interact with the C2-hydroxyl group of natural substrates, and gave up to 5-fold improvement in specific activity and 64-fold improvement in specificity towards propionaldehyde relative to glycolaldehyde. This suggests that saturation mutagenesis can be more selectively guided for evolution towards either natural or non-natural substrates, using both structural and sequence information.     

Transgenesis of schistosomes: approaches employing mobile genetic elements

Draft genome sequences for Schistosoma mansoni and Schistosoma japonicum are now available. However, the identity and importance of most schistosome genes have yet to be determined. Recently, progress has been made towards the genetic manipulation and transgenesis of schistosomes. Both loss-of-function and gain-of-function approaches appear to be feasible in schistosomes based on findings described in the past 5 years. This review focuses on reports of schistosome transgenesis, specifically those dealing with the transformation of schistosomes with exogenous mobile genetic elements and/or their endogenous relatives for the genetic manipulation of schistosomes. Transgenesis mediated by mobile genetic elements offers a potentially tractable route to introduce foreign genes to schistosomes, a means to determine the importance of schistosome genes, including those that could be targeted in novel interventions and the potential to undertake large-scale forward genetics by insertional mutagenesis.     

利用人工锌指蛋白核酸酶进行植物基因定点突变和置换

基因定点突变技术在基因组原位改变基因特定序列,避免常规转基因过程中位置效应和插入失活。定点突变生物体不含转基因或标记基因,降低风险性。高等植物基因定点突变研究初见端倪,将可能为基因原位功能研究、作物遗传改良和分子设计提供有效策略。利用锌指蛋白核酸酶（Zinc Finger Nucleases, ZFN）引入DNA定点断裂（Double-Strand Breaks, DSBs）可以高效介导基因定点突变,使得ZFN在基因定点突变中倍受关注。文章综述了植物基因定点突变的一般策略,重点介绍了锌指蛋白的结构、原理、应用,特别是ZFN介导的植物基因定点突变与置换研究进展,并对ZFN介导的植物基因定点突变与置换应用前景进行了讨论。     

A cytosine base editor toolkit with varying activity windows and target scopes for versatile gene manipulation in plants

CRISPR/Cas-derived base editing tools empower efficient alteration of genomic cytosines or adenines associated with essential genetic traits in plants and animals. Diversified target sequences and customized editing products call for base editors with distinct features regarding the editing window and target scope. Here we developed a toolkit of plant base editors containing AID10, an engineered human AID cytosine deaminase. When fused to the N-terminus or C-terminus of the conventional Cas9 nickase (nSpCas9), AID10 exhibited a broad or narrow activity window at the protospacer adjacent motif (PAM)-distal and -proximal protospacer, respectively, while AID10 fused to both termini conferred an additive activity window. We further replaced nSpCas9 with orthogonal or PAM-relaxed Cas9 variants to widen target scopes. Moreover, we devised dual base editors with AID10 located adjacently or distally to the adenine deaminase ABE8e, leading to juxtaposed or spaced cytosine and adenine co-editing at the same target sequence in plant cells. Furthermore, we expanded the application of this toolkit in plants for tunable knockdown of protein-coding genes via creating upstream open reading frame and for loss-of-function analysis of non-coding genes, such as microRNA sponges. Collectively, this toolkit increases the functional diversity and versatility of base editors in basic and applied plant research.     

A fast neutron deletion mutagenesis-based reverse genetics system for plants

A new reverse genetics method has been developed to identify and isolate deletion mutants for targeted plant genes. Deletion mutant libraries are generated using fast neutron bombardment. DNA samples extracted from the deletion libraries are used to screen for deletion mutants by polymerase chain reaction (PCR) using specific primers flanking the targeted genes. By adjusting PCR conditions to preferentially amplify the deletion alleles, deletion mutants were identified in pools of DNA samples, each pool containing DNA from 2592 mutant lines. Deletion mutants were obtained for 84% of targeted loci from an Arabidopsis population of 51 840 lines. Using a similar approach, a deletion mutant for a rice gene was identified. Thus we demonstrate that it is possible to apply this method to plant species other than Arabidopsis. As fast neutron mutagenesis is highly efficient, it is practical to develop deletion mutant populations with more complete coverage of the genome than obtained with methods based on insertional mutagenesis. Because fast neutron mutagenesis is applicable to all plant genetic systems, this method has the potential to enable reverse genetics for a wide range of plant species.