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Bacterial genome reductions: Tools, applications, and challenges. 细菌基因组缩减:工具、应用和挑战。
Pub Date : 2022-08-31 eCollection Date: 2022-01-01 DOI: 10.3389/fgeed.2022.957289
Nicole LeBlanc, Trevor C Charles

Bacterial cells are widely used to produce value-added products due to their versatility, ease of manipulation, and the abundance of genome engineering tools. However, the efficiency of producing these desired biomolecules is often hindered by the cells' own metabolism, genetic instability, and the toxicity of the product. To overcome these challenges, genome reductions have been performed, making strains with the potential of serving as chassis for downstream applications. Here we review the current technologies that enable the design and construction of such reduced-genome bacteria as well as the challenges that limit their assembly and applicability. While genomic reductions have shown improvement of many cellular characteristics, a major challenge still exists in constructing these cells efficiently and rapidly. Computational tools have been created in attempts at minimizing the time needed to design these organisms, but gaps still exist in modelling these reductions in silico. Genomic reductions are a promising avenue for improving the production of value-added products, constructing chassis cells, and for uncovering cellular function but are currently limited by their time-consuming construction methods. With improvements to and the creation of novel genome editing tools and in silico models, these approaches could be combined to expedite this process and create more streamlined and efficient cell factories.

细菌细胞因其多功能性、易于操作和丰富的基因组工程工具而被广泛用于生产增值产品。然而,产生这些所需生物分子的效率常常受到细胞自身代谢、遗传不稳定性和产物毒性的阻碍。为了克服这些挑战,已经进行了基因组缩减,使菌株具有作为下游应用的基础的潜力。在这里,我们回顾了当前能够设计和构建这种减少基因组细菌的技术,以及限制其组装和适用性的挑战。虽然基因组减少已经显示出许多细胞特征的改善,但有效和快速构建这些细胞仍然存在一个主要挑战。为了尽量减少设计这些生物所需的时间,已经创建了计算工具,但在计算机模拟这些减少方面仍然存在差距。基因组还原是提高增值产品生产、构建底盘细胞和揭示细胞功能的有希望的途径,但目前受其耗时的构建方法的限制。随着新的基因组编辑工具和计算机模型的改进和创建,这些方法可以结合起来加速这一过程,并创建更精简和高效的细胞工厂。
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引用次数: 10
Natural and artificial sources of genetic variation used in crop breeding: A baseline comparator for genome editing. 用于作物育种的天然和人工遗传变异源:基因组编辑的基准比较。
IF 4.9 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2022-08-22 eCollection Date: 2022-01-01 DOI: 10.3389/fgeed.2022.937853
Jorge Martínez-Fortún, Dylan W Phillips, Huw D Jones

Traditional breeding has successfully selected beneficial traits for food, feed, and fibre crops over the last several thousand years. The last century has seen significant technological advancements particularly in marker assisted selection and the generation of induced genetic variation, including over the last few decades, through mutation breeding, genetic modification, and genome editing. While regulatory frameworks for traditional varietal development and for genetic modification with transgenes are broadly established, those for genome editing are lacking or are still evolving in many regions. In particular, the lack of "foreign" recombinant DNA in genome edited plants and that the resulting SNPs or INDELs are indistinguishable from those seen in traditional breeding has challenged development of new legislation. Where products of genome editing and other novel breeding technologies possess no transgenes and could have been generated via traditional methods, we argue that it is logical and proportionate to apply equivalent legislative oversight that already exists for traditional breeding and novel foods. This review analyses the types and the scale of spontaneous and induced genetic variation that can be selected during traditional plant breeding activities. It provides a base line from which to judge whether genetic changes brought about by techniques of genome editing or other reverse genetic methods are indeed comparable to those routinely found using traditional methods of plant breeding.

过去几千年来,传统育种成功地为粮食、饲料和纤维作物选育出有益的性状。上个世纪,技术取得了长足进步,特别是在标记辅助选择和诱导遗传变异的产生方面,包括在过去几十年里,通过突变育种、基因改造和基因组编辑等手段。传统的品种培育和转基因的基因修饰的监管框架已广泛建立,但基因组编辑的监管框架在许多地区还缺乏或仍在发展中。特别是基因组编辑植物中缺乏 "外来 "重组 DNA,以及由此产生的 SNPs 或 INDELs 与传统育种中的 SNPs 或 INDELs 无法区分,这些都对新立法的制定提出了挑战。我们认为,如果基因组编辑和其他新型育种技术的产品不具有转基因,并且可以通过传统方法生成,那么对传统育种和新型食品适用已有的同等法律监督是合乎逻辑和适度的。本综述分析了传统植物育种活动中可选择的自发和诱导遗传变异的类型和规模。它为判断基因组编辑技术或其他反向遗传方法所带来的遗传变异是否与传统植物育种方法所发现的遗传变异具有可比性提供了一个基础。
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引用次数: 0
Corrigendum: Strategies for Efficient Gene Editing in Protoplasts of Solanum tuberosum Theme: Determining gRNA Efficiency Design by Utilizing Protoplast (Research). 主题:利用原生质体确定gRNA效率设计(研究)。
Pub Date : 2022-08-04 eCollection Date: 2022-01-01 DOI: 10.3389/fgeed.2022.914100
Frida Meijer Carlsen, Ida Elisabeth Johansen, Zhang Yang, Ying Liu, Ida Nøhr Westberg, Nam Phuong Kieu, Bodil Jørgensen, Marit Lenman, Erik Andreasson, Kåre Lehmann Nielsen, Andreas Blennow, Bent Larsen Petersen

[This corrects the article DOI: 10.3389/fgeed.2021.795644.].

[这更正了文章DOI: 10.3389/fgeed.2021.795644.]。
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引用次数: 2
Improvements of nuclease and nickase gene modification techniques for the treatment of genetic diseases. 核酸酶和镍酶基因修饰技术在遗传性疾病治疗中的改进。
Pub Date : 2022-07-26 eCollection Date: 2022-01-01 DOI: 10.3389/fgeed.2022.892769
Yaoyao Lu, Cedric Happi Mbakam, Bo Song, Eli Bendavid, Jacques-P Tremblay

Advancements in genome editing make possible to exploit the functions of enzymes for efficient DNA modifications with tremendous potential to treat human genetic diseases. Several nuclease genome editing strategies including Meganucleases (MNs), Zinc Finger Nucleases (ZFNs), Transcription Activator-like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins (CRISPR-Cas) have been developed for the correction of genetic mutations. CRISPR-Cas has further been engineered to create nickase genome editing tools including Base editors and Prime editors with much precision and efficacy. In this review, we summarized recent improvements in nuclease and nickase genome editing approaches for the treatment of genetic diseases. We also highlighted some limitations for the translation of these approaches into clinical applications.

基因组编辑的进步使得利用酶的功能进行有效的DNA修饰成为可能,这对治疗人类遗传疾病具有巨大的潜力。几种核酸酶基因组编辑策略,包括巨核酶(MNs)、锌指核酸酶(ZFNs)、转录激活物样效应核酸酶(TALENs)和聚集规则间隔短回传重复序列crispr相关蛋白(CRISPR-Cas),已被开发用于纠正基因突变。CRISPR-Cas进一步被设计用于创建包括碱基编辑器和Prime编辑器在内的镍酶基因组编辑工具,具有很高的精度和效率。在这篇综述中,我们总结了核酸酶和镍酶基因组编辑方法在治疗遗传疾病方面的最新进展。我们还强调了将这些方法转化为临床应用的一些限制。
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引用次数: 3
The trans DNA cleavage activity of Cas12a provides no detectable immunity against plasmid or phage. Cas12a的反式DNA裂解活性对质粒或噬菌体没有可检测到的免疫力。
Pub Date : 2022-07-26 eCollection Date: 2022-01-01 DOI: 10.3389/fgeed.2022.929929
Shunhang Liu, Xichen Rao, Ruiliang Zhao, Wenyuan Han

Cas12a is a type V-A CRISPR-Cas RNA-guided endonuclease. It cleaves dsDNA at specific site, and then is activated for nonspecific ssDNA cleavage in trans in vitro. The immune function of the trans activity is still unknown. To address this question, we constructed a Cas12a targeting system in Escherichia coli, where Cas12a cleaved a high-copy target plasmid to unleash the trans ssDNA cleavage activity. Then, we analyzed the effect of the Cas12a targeting on a non-target plasmid and a ssDNA phage. The results show that Cas12a efficiently eliminates target plasmid but exerts no impact on the maintenance of the non-target plasmid or plague formation efficiency of the phage. In addition, a two-spacer CRISPR array, which facilitates target plasmid depletion, still has no detectable effect on the non-target plasmid or phage either. Together, the data suggest that the trans ssDNA cleavage of Cas12a does not contribute to immunity in vivo.

Cas12a是一种V-A型CRISPR-Cas rna引导的内切酶。它在特定位点切割ssDNA,然后在体外反式激活非特异性切割ssDNA。反式活性的免疫功能尚不清楚。为了解决这个问题,我们在大肠杆菌中构建了Cas12a靶向系统,其中Cas12a切割高拷贝的靶质粒以释放反式ssDNA的切割活性。然后,我们分析了Cas12a靶向非靶质粒和ssDNA噬菌体的效果。结果表明,Cas12a能够有效地消除靶质粒,但对非靶质粒的维持和噬菌体的鼠疫形成效率没有影响。此外,双间隔CRISPR阵列有利于靶质粒的耗尽,但对非靶质粒或噬菌体也没有可检测到的影响。总之,这些数据表明,Cas12a的反式ssDNA切割在体内并不有助于免疫。
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引用次数: 3
Gene Editing to Tackle Facioscapulohumeral Muscular Dystrophy. 用基因编辑技术解决面岬肱肌营养不良症。
IF 4.9 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2022-07-15 eCollection Date: 2022-01-01 DOI: 10.3389/fgeed.2022.937879
Virginie Mariot, Julie Dumonceaux

Facioscapulohumeral dystrophy (FSHD) is a skeletal muscle disease caused by the aberrant expression of the DUX4 gene in the muscle tissue. To date, different therapeutic approaches have been proposed, targeting DUX4 at the DNA, RNA or protein levels. The recent development of the clustered regularly interspaced short-palindromic repeat (CRISPR) based technology opened new avenues of research, and FSHD is no exception. For the first time, a cure for genetic muscular diseases can be considered. Here, we describe CRISPR-based strategies that are currently being investigated for FSHD. The different approaches include the epigenome editing targeting the DUX4 gene and its promoter, gene editing targeting the polyadenylation of DUX4 using TALEN, CRISPR/cas9 or adenine base editing and the CRISPR-Cas9 genome editing for SMCHD1. We also discuss challenges facing the development of these gene editing based therapeutics.

面岬肱肌营养不良症(FSHD)是一种骨骼肌疾病,由肌肉组织中 DUX4 基因的异常表达引起。迄今为止,针对 DNA、RNA 或蛋白质水平上的 DUX4 提出了不同的治疗方法。最近,基于簇状规则间距短基因重复(CRISPR)技术的发展开辟了新的研究途径,前列腺肥大症也不例外。遗传性肌肉疾病首次有了治愈的可能。在此,我们将介绍目前正在研究的基于CRISPR的FSHD治疗策略。不同的方法包括针对 DUX4 基因及其启动子的表观基因组编辑,使用 TALEN、CRISPR/cas9 或腺嘌呤碱基编辑针对 DUX4 多腺苷酸化的基因编辑,以及针对 SMCHD1 的 CRISPR-Cas9 基因组编辑。我们还讨论了开发这些基于基因编辑的疗法所面临的挑战。
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引用次数: 0
The promise of gene editing: so close and yet so perilously far. 基因编辑的前景:如此接近,却又如此危险。
Pub Date : 2022-07-15 eCollection Date: 2022-01-01 DOI: 10.3389/fgeed.2022.974798
David J Segal
On the one hand, it is striking how the promise of genome editing is advancing. Regulatory restrictions have largely eased on genetically engineered crops that carry genome modifications that are similar to spontaneous mutations or those produced by conventional chemical or radiation-based methods (Van Vu et al., 2022). Plants produced by site-directed nuclease type 1 methods (SDN1), for which substitutions and indels are produced only by the action of the nuclease, have been deregulated in many countries. An exception are those countries within the European Union, where, despite being the third largest producer of genetically engineered crops behind China and the USA, SDN1 crops remain subject to the stringent regulations for genetically modified organisms (GMOs). Such stringent regulations are considered to have a dampening effect on agriculture innovation in the EU, and are perhaps similar to the dampening effect of long regulatory delays on the genetic engineering of livestock animals (Van Eenennaam et al., 2021). Since the first report of genetic engineering in livestock animals in 1985, only a single food animal has been commercialized. This is in part due to the USA Food and Drug Administration and their EU counterparts classifying any intentional altered genomic DNA in animals as an investigational new animal drug (INAD) that is not generally recognized as safe. However, there is a growing realization that the current EU policy towards SDN1 crops needs to be updated (Dima et al., 2022), giving hope to the wider use of these directed editing methods that can dramatically accelerate the production of new varieties compared to traditional breeding techniques. Interestingly, regulations have not hindered innovation in the application of genetic engineering to human health. In fact, this area has been a significant driver of technological advances. Recent publications and scientific meetings, such as the Keystone Symposium on Precision Genome Engineering and the American Society for Gene and Cell Therapy Annual Meeting, highlight the rapid advances in genome editing tools, driven in large part by a sense that new treatments for human disease enabled by these tools are just around the corner. Indeed, by some estimates there are over 100 products using genome editors now in clinical trial (CRISPR Medicine News), led by companies, such as CRISPR Therapeutics, Intellia Therapeutics, Sangamo Therapeutics, Editas Medicine, Precision Biosciences, Caribou Biosciences, Locus Biosciences, and many others. In the academic sector, Phase 1 of the NIH Somatic Cell Genome Editing Consortium (Saha et al., 2021), which had focused primarily on developing new editors and delivery methods, has led to a Phase 2 that is primarily focused on using these tools to OPEN ACCESS
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引用次数: 3
Examination of the Cell Cycle Dependence of Cytosine and Adenine Base Editors. 胞嘧啶和腺嘌呤碱基编辑器细胞周期依赖性的研究。
Pub Date : 2022-07-14 eCollection Date: 2022-01-01 DOI: 10.3389/fgeed.2022.923718
Cameron A Burnett, Ashley T Wong, Carlos A Vasquez, Colleen A McHugh, Gene W Yeo, Alexis C Komor

Base editors (BEs) are genome editing agents that install point mutations with high efficiency and specificity. Due to their reliance on uracil and inosine DNA damage intermediates (rather than double-strand DNA breaks, or DSBs), it has been hypothesized that BEs rely on more ubiquitous DNA repair pathways than DSB-reliant genome editing methods, which require processes that are only active during certain phases of the cell cycle. We report here the first systematic study of the cell cycle-dependence of base editing using cell synchronization experiments. We find that nickase-derived BEs (which introduce DNA backbone nicks opposite the uracil or inosine base) function independently of the cell cycle, while non-nicking BEs are highly dependent on S-phase (DNA synthesis phase). We found that synchronization in G1 (growth phase) during the process of cytosine base editing causes significant increases in C•G to A•T "byproduct" introduction rates, which can be leveraged to discover new strategies for precise C•G to A•T base editing. We observe that endogenous expression levels of DNA damage repair pathways are sufficient to process base editing intermediates into desired editing outcomes, and the process of base editing does not significantly perturb transcription levels. Overall, our study provides mechanistic data demonstrating the robustness of nickase-derived BEs for performing genome editing across the cell cycle.

碱基编辑器(BEs)是一种高效率、特异性安装点突变的基因组编辑试剂。由于它们依赖于尿嘧啶和肌苷DNA损伤中间体(而不是双链DNA断裂或dsb),因此有人假设,与依赖于dsb的基因组编辑方法相比,BEs依赖于更普遍的DNA修复途径,后者需要仅在细胞周期的某些阶段活跃的过程。我们在这里报告了使用细胞同步实验对碱基编辑的细胞周期依赖性的第一个系统研究。我们发现,缺口酶衍生的BEs(引入与尿嘧啶或肌苷碱基相反的DNA主干缺口)的功能独立于细胞周期,而非缺口的BEs高度依赖于s期(DNA合成期)。我们发现胞嘧啶碱基编辑过程中G1(生长阶段)的同步导致C•G到A•T“副产物”引入率显著增加,这可以用来发现精确的C•G到A•T碱基编辑的新策略。我们观察到DNA损伤修复途径的内源性表达水平足以将碱基编辑中间体加工成所需的编辑结果,并且碱基编辑过程不会显著干扰转录水平。总的来说,我们的研究提供了机制数据,证明了镍酶衍生的BEs在整个细胞周期中进行基因组编辑的稳健性。
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引用次数: 4
Optimizing CRISPR/Cas9 Editing of Repetitive Single Nucleotide Variants. 优化重复单核苷酸变异的CRISPR/Cas9编辑
Pub Date : 2022-07-05 eCollection Date: 2022-01-01 DOI: 10.3389/fgeed.2022.932434
Inga Usher, Lorena Ligammari, Sara Ahrabi, Emily Hepburn, Calum Connolly, Gareth L Bond, Adrienne M Flanagan, Lucia Cottone

CRISPR/Cas9, base editors and prime editors comprise the contemporary genome editing toolbox. Many studies have optimized the use of CRISPR/Cas9, as the original CRISPR genome editing system, in substituting single nucleotides by homology directed repair (HDR), although this remains challenging. Studies describing modifications that improve editing efficiency fall short of isolating clonal cell lines or have not been validated for challenging loci or cell models. We present data from 95 transfections using a colony forming and an immortalized cell line comparing the effect on editing efficiency of donor template modifications, concentration of components, HDR enhancing agents and cold shock. We found that in silico predictions of guide RNA efficiency correlated poorly withactivity in cells. Using NGS and ddPCR we detected editing efficiencies of 5-12% in the transfected populations which fell to 1% on clonal cell line isolation. Our data demonstrate the variability of CRISPR efficiency by cell model, target locus and other factors. Successful genome editing requires a comparison of systems and modifications to develop the optimal protocol for the cell model and locus. We describe the steps in this process in a flowchart for those embarking on genome editing using any system and incorporate validated HDR-boosting modifications for those using CRISPR/Cas9.

CRISPR/Cas9、碱基编辑器和初始编辑器构成了当代基因组编辑工具箱。许多研究优化了CRISPR/Cas9作为最初的CRISPR基因组编辑系统,通过同源定向修复(homology directed repair, HDR)取代单核苷酸的使用,尽管这仍然具有挑战性。描述修饰提高编辑效率的研究缺乏克隆细胞系的分离,或者尚未在具有挑战性的位点或细胞模型中得到验证。我们展示了使用集落形成和永生化细胞系进行的95次转染的数据,比较了供体模板修饰、成分浓度、HDR增强剂和冷休克对编辑效率的影响。我们发现引导RNA效率的计算机预测与细胞中的活性相关性很差。使用NGS和ddPCR,我们检测到在转染的群体中编辑效率为5-12%,在克隆细胞系分离时下降到1%。我们的数据证明了细胞模型、靶基因座和其他因素对CRISPR效率的影响。成功的基因组编辑需要系统和修改的比较,以制定细胞模型和位点的最佳方案。我们在流程图中描述了这个过程中的步骤,适用于那些使用任何系统进行基因组编辑的人,并为使用CRISPR/Cas9的人整合了经过验证的hdr增强修饰。
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引用次数: 2
Invited Mini-Review Research Topic: Utilization of Protoplasts to Facilitate Gene Editing in Plants: Schemes for In Vitro Shoot Regeneration From Tissues and Protoplasts of Potato and Rapeseed: Implications of Bioengineering Such as Gene Editing of Broad-Leaved Plants. 研究主题:利用原生质体促进植物基因编辑;马铃薯和油菜籽组织和原生质体离体芽再生方案;阔叶植物基因编辑等生物工程的意义。
Pub Date : 2022-06-29 eCollection Date: 2022-01-01 DOI: 10.3389/fgeed.2022.780004
Erik Andreasson, Nam Phuong Kieu, Muhammad Awais Zahid, Frida Meijer Carlsen, Lenman Marit, Sjur Sandgrind, Bent Larsen Petersen, Li-Hua Zhu

Schemes for efficient regenerationand recovery of shoots from in vitro tissues or single cells, such as protoplasts, are only available for limited numbers of plant species and genotypes and are crucial for establishing gene editing tools on a broader scale in agriculture and plant biology. Growth conditions, including hormone and nutrient composition as well as light regimes in key steps of known regeneration protocols, display significant variations, even between the genotypes within the same species, e.g., potato (Solanum tuberosum). As fresh plant material is a prerequisite for successful shoot regeneration, the plant material often needs to be refreshed for optimizing the growth and physiological state prior to genetic transformation. Utilization of protoplasts has become a more important approach for obtaining transgene-free edited plants by genome editing, CRISPR/Cas9. In this approach, callus formation from protoplasts is induced by one set of hormones, followed by organogenesis, i.e., shoot formation, which is induced by a second set of hormones. The requirements on culture conditions at these key steps vary considerably between the species and genotypes, which often require quantitative adjustments of medium compositions. In this mini-review, we outline the protocols and notes for clonal regeneration and cultivation from single cells, particularly protoplasts in potato and rapeseed. We focus mainly on different hormone treatment schemes and highlight the importance of medium compositions, e.g., sugar, nutrient, and light regimes as well as culture durations at the key regeneration steps. We believe that this review would provide important information and hints for establishing efficient regeneration strategies from other closely related and broad-leaved plant species in general.

从离体组织或单细胞(如原生质体)中高效再生和恢复芽的方案仅适用于有限数量的植物物种和基因型,对于在农业和植物生物学中建立更大规模的基因编辑工具至关重要。生长条件,包括激素和营养成分以及已知再生方案关键步骤的光照制度,显示出显著的差异,甚至在同一物种的基因型之间,例如马铃薯(Solanum tuberosum)。由于新鲜的植株材料是茎部再生成功的先决条件,在遗传转化之前,经常需要对植株材料进行更新,以优化其生长和生理状态。利用原生质体已成为基因组编辑CRISPR/Cas9获得无转基因编辑植物的更重要途径。在这种方法中,原生质体的愈伤组织形成是由一组激素诱导的,然后是器官发生,即由第二组激素诱导的嫩枝形成。这些关键步骤对培养条件的要求在物种和基因型之间有很大差异,这通常需要对培养基成分进行定量调整。在这篇综述中,我们概述了单细胞克隆再生和培养的方案和注意事项,特别是马铃薯和油菜籽的原生质体。我们主要关注不同的激素处理方案,并强调培养基组成的重要性,例如糖、营养物质、光照制度以及关键再生步骤的培养时间。我们相信这一综述将为其他近缘阔叶植物建立有效的更新策略提供重要的信息和提示。
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引用次数: 1
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Frontiers in genome editing
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