CRISPR/Cas9介导烟草多基因编辑体系的应用

CRISPR/Cas9是一种新型的基因组定向编辑技术,近年来在烟草基因组的定向编辑中得到了广泛应用。CRISPR/Cas9介导的多基因编辑技术在许多物种中表现出很大的潜力,为了探索CRISPR/Cas9介导烟草多基因编辑的技术体系,本文针对烟草PCS1、HMA2、eIF4E、TOM3、TOM1 5个不同性状相关的基因构建了多靶点敲除的CRISPR/Cas9系统,并借助农杆菌介导的遗传转化技术转化烟草品种K326。对阳性植株的筛选、靶位点基因组的PCR扩增与测序分析表明,构建的CRISPR/Cas9多基因编辑系统成功转化到烟草中,能够同时靶向突变5个基因。5个基因同时突变的检出率为53.8%,单基因的突变检出率在76.9%与92.3%之间。进一步的脱靶检测分析显示,在所预测脱靶的候选位点上均未发生脱靶现象。本文构建的CRISPR/Cas9多基因编辑系统可将多个基因进行有效突变,为烟草基因功能研究和基于CRISPR/Cas9技术的多性状改良奠定了基础。      

CRISPR/Cpf1 enables fast and simple genome editing of Saccharomyces cerevisiae

Cpf1 represents a novel single RNA‐guided CRISPR/Cas endonuclease system suitable for genome editing with distinct features compared with Cas9. We demonstrate the functionality of three Cpf1 orthologues – Acidaminococcus spp. BV3L6 (AsCpf1), Lachnospiraceae bacterium ND2006 (LbCpf1) and Francisella novicida U112 (FnCpf1) – for genome editing of Saccharomyces cerevisiae. These Cpf1‐based systems enable fast and reliable introduction of donor DNA on the genome using a two‐plasmid‐based editing approach together with linear donor DNA. LbCpf1 and FnCpf1 displayed editing efficiencies comparable with the CRISPR/Cas9 system, whereas AsCpf1 editing efficiency was lower. Further characterization showed that AsCpf1 and LbCpf1 displayed a preference for their cognate crRNA, while FnCpf1‐mediated editing with similar efficiencies was observed using non‐cognate crRNAs of AsCpf1 and LbCpf1. In addition, multiplex genome editing using a single LbCpf1 crRNA array is shown to be functional in yeast. This work demonstrates that Cpf1 broadens the genome editing toolbox available for Saccharomyces cerevisiae. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.     

New vectors for simple and streamlined CRISPR–Cas9 genome editing in Saccharomyces cerevisiae

Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 technology is an important tool for genome editing because the Cas9 endonuclease can induce targeted DNA double‐strand breaks. Targeting of the DNA break is typically controlled by a single‐guide RNA (sgRNA), a chimeric RNA containing a structural segment important for Cas9 binding and a 20mer guide sequence that hybridizes to the genomic DNA target. Previous studies have demonstrated that CRISPR–Cas9 technology can be used for efficient, marker‐free genome editing in Saccharomyces cerevisiae. However, introducing the 20mer guide sequence into yeast sgRNA expression vectors often requires cloning procedures that are complex, time‐consuming and/or expensive. To simplify this process, we have developed a new sgRNA expression cassette with internal restriction enzyme sites that permit rapid, directional cloning of 20mer guide sequences. Here we describe a flexible set of vectors based on this design for cloning and expressing sgRNAs (and Cas9) in yeast using different selectable markers. We anticipate that the Cas9–sgRNA expression vector with the URA3 selectable marker (pML104) will be particularly useful for genome editing in yeast, since the Cas9 machinery can be easily removed by counter‐selection using 5‐fluoro‐orotic acid (5‐FOA) following successful genome editing. The availability of new vectors that simplify and streamline the technical steps required for guide sequence cloning should help accelerate the use of CRISPR–Cas9 technology in yeast genome editing. Vectors pT040, pJH001, pML104 and pML107 have been deposited at Addgene ( www.addgene.org ). Copyright © 2015 John Wiley & Sons, Ltd.     

History of genome editing in yeast

For thousands of years humans have used the budding yeast Saccharomyces cerevisiae for the production of bread and alcohol; however, in the last 30–40 years our understanding of the yeast biology has dramatically increased, enabling us to modify its genome. Although S. cerevisiae has been the main focus of many research groups, other non‐conventional yeasts have also been studied and exploited for biotechnological purposes. Our experiments and knowledge have evolved from recombination to high‐throughput PCR‐based transformations to highly accurate CRISPR methods in order to alter yeast traits for either research or industrial purposes. Since the release of the genome sequence of S. cerevisiae in 1996, the precise and targeted genome editing has increased significantly. In this ‘Budding topic’ we discuss the significant developments of genome editing in yeast, mainly focusing on Cre‐loxP mediated recombination, delitto perfetto and CRISPR/Cas.     

A history of genome editing in Saccharomyces cerevisiae

Genome editing is a form of highly precise genetic engineering which produces alterations to an organism's genome as small as a single base pair with no incidental or auxiliary modifications; this technique is crucial to the field of synthetic biology, which requires such precision in the installation of novel genetic circuits into host genomes. While a new methodology for most organisms, genome editing capabilities have been used in the budding yeast Saccharomyces cerevisiae for decades. In this review, I will present a brief history of genome editing in S. cerevisiae, discuss the current gold standard method of Cas9‐mediated genome editing, and speculate on future directions of the field.     

基于CRISPR/Cas9技术的烟草烟碱相关基因敲除及功能研究

CRISPR/Cas9是一种重要的基因组定向编辑技术,近年来在植物基因组的定向敲除和分子育种材料创制中得到了广泛应用。为了发掘可用于分子育种的低烟碱烟草,以烤烟品种K326为试验材料,利用CRISPR/Cas9基因组编辑技术对控制烟草烟碱合成和转运的5个基因（PMT1、QPT1、A622、NtNUP1和JAT1）进行定向敲除,并对T2代纯合基因型烟草植株进行烟碱含量检测。结果表明,定向敲除5个基因后获得100株成功编辑的T0代烟草植株,突变检出率为29.9%,突变类型以单碱基插入或删除为主。对定向敲除烟碱合成基因QPT1和转运基因JAT1的T1代纯合基因型烟草植株进行序列分析,发现存在4种突变类型,分别是插入一个T、C、A碱基的插入突变和一个缺失44个碱基的缺失突变,从而引发移码使得翻译的氨基酸链大幅缩短,其T2代植株上部叶烟碱含量显著低于野生型植株。综上所述,CRISPR/Cas9技术能够高效定向敲除烟碱关键基因,为低烟碱烟草分子育种提供了理论和技术支撑。     

CRISPR/Cas9-RNA interference system for combinatorial metabolic engineering of Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is widely used in industrial biotechnology for the production of fuels, chemicals, food ingredients, food and beverages, and pharmaceuticals. To obtain high-performing strains for such bioprocesses, it is often necessary to test tens or even hundreds of metabolic engineering targets, preferably in combinations, to account for synergistic and antagonistic effects. Here, we present a method that allows simultaneous perturbation of multiple selected genetic targets by combining the advantage of CRISPR/Cas9, in vivo recombination, USER assembly and RNA interference. CRISPR/Cas9 introduces a double-strand break in a specific genomic region, where multiexpression constructs combined with the knockdown constructs are simultaneously integrated by homologous recombination. We show the applicability of the method by improving cis,cis-muconic acid production in S. cerevisiae through simultaneous manipulation of several metabolic engineering targets. The method can accelerate metabolic engineering efforts for the construction of future cell factories.     

烟草单倍体基因编辑系统的建立

  背景和目的  单倍体材料作为转化受体具有不受同源染色体联会配对影响、转化效率较高、外源基因在后代基因组中稳定性较好、加倍后即可获得纯合转化植株等优点,结合基因编辑CRISPR/Cas9系统,可大大加快烟草品种选育进程。  方法  通过花粉离体培育获得单倍体,利用CRISPR/Cas9系统编辑八氢番茄红素脱氢酶基因位点,产生白化单倍体植株。  结果  （1）经过4℃处理4~6天的实验组,花药愈伤组织形成率最高可达11%;（2）获得转化后单倍体植株120株,其中白化86株（白化率71.67%）,红花大金元51株,其中白化24株（白化率47.06%）。  结论  （1）适当低温处理可提高花药培育单倍体效率;（2）以单倍体为受体的基因编辑效率有所提高。      

基于细胞融合开发双基因共敲除技术

基因编码工具(例如CRISPR/Cas9)的出现使敲除哺乳动物细胞基因成为现实.然而,实现细胞的多基因敲除成功率低且所需时间长.细胞融合技术是构建杂合细胞的常用方法,通过融合两种不同表型的细胞,构建出一种具有杂合表型的新型细胞.但是,由于基因的互补作用,通过基因敲除而获得的性状在细胞融合后成为隐性性状,融合细胞不能表现...     

Use of a fluoride channel as a new selection marker for fission yeast plasmids and application to fast genome editing with CRISPR/Cas9

Fission yeast is a powerful model organism that has provided insights into important cellular processes thanks to the ease of its genome editing by homologous recombination. However, creation of strains with a large number of targeted mutations or containing plasmids has been challenging because only a very small number of selection markers is available in Schizosaccharomyces pombe. In this paper, we identify two fission yeast fluoride exporter channels (Fex1p and Fex2p) and describe the development of a new strategy using Fex1p as a selection marker for transformants in rich media supplemented with fluoride. To our knowledge this is the first positive selection marker identified in S. pombe that does not use auxotrophy or drug resistance and that can be used for plasmids transformation or genomic integration in rich media. We illustrate the application of our new marker by significantly accelerating the protocol for genome edition using CRISPR/Cas9 in S. pombe. Copyright © 2016 John Wiley & Sons, Ltd.     

CRISPR-Cas系统在食品微生物中的应用研究进展

成簇的规律间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)及其关联蛋白(CRISPR-associated proteins,Cas)构成CRISPR-Cas系统,该系统作为高效、灵活、易于操作的基因编辑技术,开始广泛应用于微生物基因组位点的靶向编辑中。本文针对CRISPR-Cas9系统在食品微生物领域,特别是在食品酿造微生物和食品病原微生物中的应用进行综述,并进一步讨论影响该系统基因编辑效率的主要因素及发展方向,为CRISPR-Cas9系统在食品微生物中的应用提供参考和依据。     

CRISPR核酸检测技术的研究进展

作为一种新兴的基因组编辑和调控工具,CRISPR技术的诞生和发展极大地改变了生物学领域的前进方向。近几年来,CRISPR系统及其相关蛋白(Cas效应蛋白)的工作原理被逐渐揭示,由于其优越的灵敏度和特异性,该技术在多个领域开始发挥至关重要的作用。借助其新颖的核酸酶活性,人们打开了研发核酸检测新方法的大门。本文总结了多种CRISPR/ Cas系统及其在核酸检测领域的应用和局限性,并对其后续发展方向进行了展望。随着该技术的不断成熟,CRISPR/ Cas系统将进一步推动基础生物学和应用生物学的研究,并有潜力成为下一代诊断生物传感平台的候选者。     

CRISPR/Cas系统在食品质量安全检测中的应用研究进展

规律间隔成簇短回文重复序列(regularly spaced clustered short palindrome repeats-associated, CRISPR/Cas)系统是基于原核生物的一种适应性免疫系统, 因其可编程性和易操作的优点已被开发为基因编辑技术, 并且凭借其快速和高效的特点被广泛用于生物、医学等领域。目前, CRISPR/Cas系统已开始在基于核酸检测和单核苷酸多态性检测等食品安全检测技术中应用。本文就CRISPR/Cas系统中Cas9、Cas12a和Cas13a的原理以及其在掺假检测、溯源检测、食源性微生物和动物疫病等食品质量安全检测中的应用进展进行了综述, 以期为CRISPR/Cas系统应用于食品质量安全的快速现场检测提供理论和技术参考。     

基于CRISPR/Cas9系统对乳酸乳球菌进行绿色荧光蛋白标记

乳酸乳球菌是治疗剂在体内运送的良好载体,研究其在体内的真实运送情况需对其进行标记。该实验利用CRISPR/Cas9系统对乳酸乳球菌(Lactococcus lactis)NZ9000进行增强型绿色荧光蛋白(enhanced green fluorescent protein, eGFP)标记,用于研究乳酸乳球菌在体内的运送,评价其作为益生菌的功能。基于该实验室已构建的乳酸乳球菌CRISPR/Cas9编辑质粒pLL25构建重组质粒pYSH,其上携带eGFP及同源臂,电转入乳酸乳球菌NZ9000感受态细胞中,将基因组中的乳酸脱氢酶基因(ldh)替换为绿色荧光蛋白基因,从而使Lactococcus lactis NZ9000获得标记,表达绿色荧光蛋白。对绿色荧光标记的Lactococcus lactis NZ9000突变株,酶标仪定量分析eGFP的表达强度。荧光强度定量分析结果表明,在乳酸乳球菌不同生长阶段,eGFP基因均能稳定表达。     

基于CRISPR/Cas9系统对乳酸乳球菌进行绿色荧光蛋白标记

乳酸乳球菌是治疗剂在体内运送的良好载体,研究其在体内的真实运送情况需对其进行标记。该实验利用CRISPR/Cas9系统对乳酸乳球菌(Lactococcus lactis)NZ9000进行增强型绿色荧光蛋白(enhanced green fluorescent protein, eGFP)标记,用于研究乳酸乳球菌在体内的运送,评价其作为益生菌的功能。基于该实验室已构建的乳酸乳球菌CRISPR/Cas9编辑质粒pLL25构建重组质粒pYSH,其上携带eGFP及同源臂,电转入乳酸乳球菌NZ9000感受态细胞中,将基因组中的乳酸脱氢酶基因(ldh)替换为绿色荧光蛋白基因,从而使Lactococcus lactis NZ9000获得标记,表达绿色荧光蛋白。对绿色荧光标记的Lactococcus lactis NZ9000突变株,酶标仪定量分析eGFP的表达强度。荧光强度定量分析结果表明,在乳酸乳球菌不同生长阶段,eGFP基因均能稳定表达。     

基于CRISPR/Cas9系统的金针菇基因组编辑载体构建

采用高效基因编辑系统CRISPR/Cas9,构建金针菇基因Mads-8敲除载体及转化体系。以转录组数据为依据,通过分析基因Mads-8序列信息,选择高效的靶位点序列,同时加入筛选标记基因hph构建过渡载体sgRNA-T,通过菌落PCR鉴定和测序来验证靶位点序列是否正确插入。以表达载体pg Fvs-cas9为框架,酶切连接后构建重组敲除载体pg Fvs-Cas9-gRNA,PCR和酶切鉴定敲除载体。再以PEG介导的金针菇原生质体转化表达载体,在潮霉素抗性平板上筛选拟转化子,以拟转化子基因组DNA为模板扩增Cas9基因。结果显示,靶位点序列成功插入敲除载体且序列正确,重组质粒成功转化进金针菇的基因组。对金针菇基因组敲除载体的构建,为后续金针菇相关基因的功能验证及育种研究提供载体材料及理论依据。      

新一代诊断技术：CRISPR系统及其在分子诊断中的应用

作为新兴的基因编辑和调控工具,CRISPR系统具有高度的特异性、灵敏度和灵活性。除在基因编辑和改造方面有重要作用外,在传染病诊断、环境监测、食品安全检测等方面也有着良好的前景。随着CRISPR/Cas系统的深入研究,通过其高特异性识别目标序列和反式切割能力,已经建立了多种简便、灵敏的生物传感平台,解决了传统方法在操作、灵敏度、特异性及检测周期方面存在的不足。作者将主要介绍应用最广的两类CRISPR/Cas系统的特征和机制,总结了基于CRISPR/Cas系统开发的核酸、蛋白质和小分子物质构建的生物传感体系和未来发展方向。     

CRISPR‐Based Technologies and the Future of Food Science

The on‐going CRISPR craze is focused on the use of Cas9‐based technologies for genome editing applications in eukaryotes, with high potential for translational medicine and next‐generation gene therapy. Nevertheless, CRISPR‐Cas systems actually provide adaptive immunity in bacteria, and have much promise for various applications in food bacteria that include high‐resolution typing of pathogens, vaccination of starter cultures against phages, and the genesis of programmable and specific antibiotics that can selectively modulate bacterial population composition. Indeed, the molecular machinery from these DNA‐encoded, RNA‐mediated, DNA‐targeting systems can be harnessed in native hosts, or repurposed in engineered systems for a plethora of applications that can be implemented in all organisms relevant to the food chain, including agricultural crops trait‐enhancement, livestock breeding, and fermentation‐based manufacturing, and for the genesis of next‐generation food products with enhanced quality and health‐promoting functionalities. CRISPR‐based applications are now poised to revolutionize many fields within food science, from farm to fork. In this review, we describe CRISPR‐Cas systems and highlight their potential for the development of enhanced foods.     

Construction and evaluation of gRNA arrays for multiplex CRISPR-Cas9

Endonuclease system CRISPR-Cas9 represents a powerful toolbox for the budding yeast's Saccharomyces cerevisiae genome perturbation. The resulting double-strand breaks are preferentially repaired via highly efficient homologous recombination, which subsequently leads to marker-free genome editing. The goal of this study was to evaluate precise targeting of multiple loci simultaneously. To construct an array of independently expressing guide RNAs (gRNAs), the genes encoding them were assembled through a BioBrick construction procedure. We designed a multiplex CRISPR-Cas9 system for targeting 6 marker genes, whereby the gRNA array was expressed from a single plasmid. To evaluate the performance of the gRNA array, the activity of the designed system was assessed by the success rate of the introduction of perturbations within the target loci: successful gRNA expression, followed by target DNA double-strand breaks formation and their repair by homologous recombination led to premature termination of the coding sequence of the marker genes, resulting in the prevention of growth of the transformants on the corresponding selection media. In conclusion, we successfully introduced up to five simultaneous perturbations within single cells of yeast S. cerevisiae using the multiplex CRISPR-Cas9 system. While this has been done before, we here present an alternative sequential BioBrick assembly with the capability to accommodate many highly similar gRNA-expression cassettes, and an exhaustive evaluation of their performance.