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 CRISPR/Cas9 method to generate heterozygous alleles in Saccharomyces cerevisiae

Saccharomyces cerevisiae is a genetically facile organism, yet multiple CRISPR/Cas9 techniques are widely used to edit its genome more efficiently and cost effectively than conventional methods. The absence of selective markers makes CRISPR/Cas9 editing particularly useful when making mutations within genes or regulatory sequences. Heterozygous mutations within genes frequently arise in the winners of evolution experiments. The genetic dissection of heterozygous alleles can be important to understanding gene structure and function. Unfortunately, the high efficiency of genome cutting and repair makes the introduction of heterozygous alleles by standard CRISPR/Cas9 technique impossible. To be able to quickly and reliably determine the individual phenotypes of the thousands of heterozygous mutations that can occur during directed evolutions is of particular interest to industrial strain improvement research. In this report, we describe a CRISPR/Cas9 method that introduces specific heterozygous mutations into the S. cerevisiae genome. This method relies upon creating silent point mutations in the protospacer adjacent motif site or removing the protospacer adjacent motif site entirely to stop the multiple rounds of genome editing that prevent heterozygous alleles from being generated. This technique should be able to create heterozygous alleles in other diploid yeasts and different allelic copy numbers in polyploid cells.     

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.     

CRISPRi/ddCpf1促进大肠杆菌中丙二酰辅酶A的积累

丙二酰辅酶A是多种有价值化合物（包括食品、保健品等）合成的重要前体物质，其合成已成为大肠杆菌中生产目标代谢物的潜在瓶颈。为解决其生物合成的瓶颈问题，作者通过CRISPR干扰（CRISPR interference，CRISPRi） 促进丙二酰辅酶A的积累。首先，构建了丙二酰辅酶A的生物传感器，实现了其快速、可视化的胞内测量。随后，构建了CRISPRi/ddCpf1系统，实现了基因转录的抑制，并尝试用ddCpf1（催化失活的Cpf1）和RNA聚合酶抑制蛋白Gp2融合表达（CRISPRi/ddCpf1-Gp2）。结果表明，Gp2虽然有一定的转录抑制效果，但是严重影响了生长。最后，利用CRISPRi/ddCpf1系统进行两组双靶点基因抑制，分别靶向乙醛脱氢酶和3-氧代酰基-酰基载体蛋白合酶II基因，以及3-氧代酰基-酰基载体蛋白合酶I和琥珀酰辅酶A合成酶基因，均实现了丙二酰辅酶A浓度的提高。     

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.     

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/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‐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.     

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,融合细胞不能表现该性状。本研究的目的是设计一种通过细胞融合技术和CRISPR/Cas9技术获得基因双敲除细胞的方法。由于现阶段没有关于HEK 293细胞株细胞融合的参考数据,所以首先在HEK 293野生型细胞株中分别敲除GPI生物合成必需的PIGA或PIGK,获得PIGA-KO细胞株和PIGK-KO细胞株,并以这两个细胞株作为模型进行条件优化。经过反应条件优化,HEK 293细胞的融合效率显著提高,且实现了FUT8敲除细胞株和ST6GAL1敲除细胞株的快速融合;其次是将细胞融合技术与CRISPR/Cas9技术结合从而实现多种糖基因的快速敲除。将分别含有FUT8和ST6GAL1目标导向RNAs且都能稳定表达Cas9蛋白的两个细胞株进行融合,即可获得FUT8和ST6GAL1基因都被敲除的双敲细胞株。实验结果表明,将细胞融合技术和CRISPR/Cas9技术结合可简便而快速地获得基因双敲除细胞株。     

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。     

大肠杆菌中吡咯喹啉醌合成途径的构建

目前吡咯喹啉醌（Pyrroloquinoline quinone,PQQ）在大肠杆菌中的异源合成主要以质粒为表达载体进行,但是质粒载体难以进行合成途径多基因表达的系统优化,并且容易造成发酵不稳定。作者以大肠杆菌为底盘生物,利用CRISPR/Cas9基因编辑技术,在基因组水平系统优化PQQ的合成。将来源于Gluconobacter oxydans 621H的操纵子pqqABCDE引入底盘大肠杆菌,并进一步通过优化合成途径基因表达强度,敲除大肠杆菌自身抑制基因及强化PQQ的胞内需求与胞外转运等,获得了一株能够高效合成PQQ的工程菌,摇瓶发酵72 h时产量达到86.3 mg/L。以大肠杆菌为底盘构建PQQ高效合成途径的工作能够为后续以其他底盘生物生产PQQ及相关代谢产物提供借鉴。     

The 50:50 method for PCR‐based seamless genome editing in yeast

The ability to edit the yeast genome with relative ease has contributed to the organism being a model eukaryote for decades. Most methods for deleting, inserting or altering genomic sequences require transformation with DNA that carries the desired change and a selectable marker. One‐step genome editing methods retain the selectable marker. Seamless genome editing methods require more steps and a marker that can be used for both positive and negative selection, such as URA3. Here we describe the PCR‐based 50:50 method for seamless genome editing, which requires only two primers, one PCR with a URA3 cassette, and a single yeast transformation. Our method is based on pop‐in/pop‐out gene replacement and is amenable to the facile creation of genomic deletions and short insertions or substitutions. We used the 50:50 method to make two conservative loss‐of‐function mutations in MATα1, with results suggesting that the wild‐type gene has a new function outside of that presently known. Copyright © 2013 John Wiley & Sons, Ltd.     

A Candida albicans gene encoding a DNA ligase

A DNA ligase-encoding gene (Ca CDC9) was cloned from Candida albicans by complementation of an ime-1 mutation in Saccharomyces cerevisiae. In this system, IME1 function was assayed using a S. cerevisiae strain with a ime2-promoter-lacZ gene fusion such that following transformation with a C. albicans genomic library, the presence of positive clones was indicated upon the addition of X-gal to sporulation media. Transforming fragments were subcloned in pGEM7 and sequenced. Sequence homology with several ATP-dependent DNA ligases from viruses, fission yeast, human, baker yeast and bacteria was observed. The sequence has been deposited in the EMBL data bank under the Accession Number X95001.     

A new inducible CRISPR-Cas9 system useful for genome editing and study of double-strand break repair in Candida glabrata

In recent years, the CRISPR-Cas9 system has proven extremely useful for genome editing in many species, including the model yeast Saccharomyces cerevisiae and other yeast species such as Candida glabrata. Inducible CRISPR-Cas9 systems have the additional advantage of allowing to separate the transformation step of the organism by the CRISPR-Cas9 system, from the cutting and repair steps. This has indeed been developed in S. cerevisiae, where most inducible expression systems rely on the GAL promoters. Unfortunately, C. glabrata is gal⁻ and lacks the GAL genes, like many other yeast species. We report here the use of a vector expressing cas9 under the control of the MET3 promoter, with the guide RNA cloned into the same plasmid. We show that it can be used efficiently in C. glabrata, for both described outcomes of CRISPR-Cas9-induced chromosome breaks; nonhomologous end joining in the absence of a homologous repair template; and homologous recombination in the presence of such a template. This system therefore allows easy editing of the genome of C. glabrata, and its inducibility may allow identification of essential genes in this asexual yeast, where spore lethality cannot be observed, as well as the study of double-strand break repair.     

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。