Frontiers in genome editing最新文献

{"title":"Bacterial genome reductions: Tools, applications, and challenges.","authors":"Nicole LeBlanc,&nbsp;Trevor C Charles","doi":"10.3389/fgeed.2022.957289","DOIUrl":"https://doi.org/10.3389/fgeed.2022.957289","url":null,"abstract":"<p><p>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 <i>in silico</i>. 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 <i>in silico</i> models, these approaches could be combined to expedite this process and create more streamlined and efficient cell factories.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9473318/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40366445","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Natural and artificial sources of genetic variation used in crop breeding: A baseline comparator for genome editing.","authors":"Jorge Martínez-Fortún, Dylan W Phillips, Huw D Jones","doi":"10.3389/fgeed.2022.937853","DOIUrl":"10.3389/fgeed.2022.937853","url":null,"abstract":"<p><p>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 <i>via</i> 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.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":null,"pages":null},"PeriodicalIF":4.9,"publicationDate":"2022-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9441798/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33448842","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Corrigendum: Strategies for Efficient Gene Editing in Protoplasts of <i>Solanum tuberosum</i> Theme: Determining gRNA Efficiency Design by Utilizing Protoplast (Research).","authors":"Frida Meijer Carlsen,&nbsp;Ida Elisabeth Johansen,&nbsp;Zhang Yang,&nbsp;Ying Liu,&nbsp;Ida Nøhr Westberg,&nbsp;Nam Phuong Kieu,&nbsp;Bodil Jørgensen,&nbsp;Marit Lenman,&nbsp;Erik Andreasson,&nbsp;Kåre Lehmann Nielsen,&nbsp;Andreas Blennow,&nbsp;Bent Larsen Petersen","doi":"10.3389/fgeed.2022.914100","DOIUrl":"https://doi.org/10.3389/fgeed.2022.914100","url":null,"abstract":"<p><p>[This corrects the article DOI: 10.3389/fgeed.2021.795644.].</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-08-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9385982/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40432120","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Improvements of nuclease and nickase gene modification techniques for the treatment of genetic diseases.","authors":"Yaoyao Lu,&nbsp;Cedric Happi Mbakam,&nbsp;Bo Song,&nbsp;Eli Bendavid,&nbsp;Jacques-P Tremblay","doi":"10.3389/fgeed.2022.892769","DOIUrl":"https://doi.org/10.3389/fgeed.2022.892769","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9360573/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40716019","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The trans DNA cleavage activity of Cas12a provides no detectable immunity against plasmid or phage.","authors":"Shunhang Liu,&nbsp;Xichen Rao,&nbsp;Ruiliang Zhao,&nbsp;Wenyuan Han","doi":"10.3389/fgeed.2022.929929","DOIUrl":"https://doi.org/10.3389/fgeed.2022.929929","url":null,"abstract":"<p><p>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 <i>in vitro</i>. The immune function of the trans activity is still unknown. To address this question, we constructed a Cas12a targeting system in <i>Escherichia coli</i>, 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 <i>in vivo</i>.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9360544/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40691087","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Gene Editing to Tackle Facioscapulohumeral Muscular Dystrophy.","authors":"Virginie Mariot, Julie Dumonceaux","doi":"10.3389/fgeed.2022.937879","DOIUrl":"10.3389/fgeed.2022.937879","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":null,"pages":null},"PeriodicalIF":4.9,"publicationDate":"2022-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9334676/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40670894","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The promise of gene editing: so close and yet so perilously far.","authors":"David J Segal","doi":"10.3389/fgeed.2022.974798","DOIUrl":"https://doi.org/10.3389/fgeed.2022.974798","url":null,"abstract":"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","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9334663/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40589852","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Examination of the Cell Cycle Dependence of Cytosine and Adenine Base Editors.","authors":"Cameron A Burnett,&nbsp;Ashley T Wong,&nbsp;Carlos A Vasquez,&nbsp;Colleen A McHugh,&nbsp;Gene W Yeo,&nbsp;Alexis C Komor","doi":"10.3389/fgeed.2022.923718","DOIUrl":"https://doi.org/10.3389/fgeed.2022.923718","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9333457/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40670895","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimizing CRISPR/Cas9 Editing of Repetitive Single Nucleotide Variants.","authors":"Inga Usher,&nbsp;Lorena Ligammari,&nbsp;Sara Ahrabi,&nbsp;Emily Hepburn,&nbsp;Calum Connolly,&nbsp;Gareth L Bond,&nbsp;Adrienne M Flanagan,&nbsp;Lucia Cottone","doi":"10.3389/fgeed.2022.932434","DOIUrl":"https://doi.org/10.3389/fgeed.2022.932434","url":null,"abstract":"<p><p>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 <i>in silico</i> 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.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9294353/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40527440","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Invited Mini-Review Research Topic: Utilization of Protoplasts to Facilitate Gene Editing in Plants: Schemes for <i>In Vitro</i> Shoot Regeneration From Tissues and Protoplasts of Potato and Rapeseed: Implications of Bioengineering Such as Gene Editing of Broad-Leaved Plants.","authors":"Erik Andreasson,&nbsp;Nam Phuong Kieu,&nbsp;Muhammad Awais Zahid,&nbsp;Frida Meijer Carlsen,&nbsp;Lenman Marit,&nbsp;Sjur Sandgrind,&nbsp;Bent Larsen Petersen,&nbsp;Li-Hua Zhu","doi":"10.3389/fgeed.2022.780004","DOIUrl":"https://doi.org/10.3389/fgeed.2022.780004","url":null,"abstract":"<p><p>Schemes for efficient regenerationand recovery of shoots from <i>in vitro</i> 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 (<i>Solanum tuberosum</i>). 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.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9276966/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40513271","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}