Frontiers in genome editing最新文献

{"title":"Genetic manipulation of bacteriophage T4 utilizing the CRISPR-Cas13b system.","authors":"Yuvaraj Bhoobalan-Chitty, Mathieu Stouf, Marianne De Paepe","doi":"10.3389/fgeed.2024.1495968","DOIUrl":"10.3389/fgeed.2024.1495968","url":null,"abstract":"<p><p>CRISPR-Cas type II and type V systems are inefficient in modifying bacteriophage T4 genome, due to hypermodification of its DNA. Here, we present a genome editing technique for bacteriophage T4 using the type VI CRISPR-Cas system. Using BzCas13b targeting of T4 phage, we were able to individually delete both T4 glucosyl transferase genes, <i>α-gt</i> and <i>β-gt</i>. Furthermore, we employed this method to mutate two conserved residues within the T4 DNA polymerase and to introduce the yellow fluorescent protein (YFP) coding sequence into T4 phage genome, enabling us to visualize phage infections. This T4 genome editing protocol was optimized to generate recombinant phages within a 6-hour timescale. Finally, spacers homologous to a variety of T4 genes were used to study the generality of Cas13b targeting, revealing important variability in targeting efficiency. Overall, this method constitutes a rapid and effective means of generating specific T4 phage mutants, which could be extended to other T4-like phages.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"6 ","pages":"1495968"},"PeriodicalIF":4.9,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11693715/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142923861","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":"Advances in CRISPR-Cas technology and its applications: revolutionising precision medicine.","authors":"Sarkar Sardar Azeez, Rahin Shareef Hamad, Bahra Kakamin Hamad, Mudhir Sabir Shekha, Peter Bergsten","doi":"10.3389/fgeed.2024.1509924","DOIUrl":"10.3389/fgeed.2024.1509924","url":null,"abstract":"<p><p>CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated proteins) has undergone marked advancements since its discovery as an adaptive immune system in bacteria and archaea, emerged as a potent gene-editing tool after the successful engineering of its synthetic guide RNA (sgRNA) toward the targeting of specific DNA sequences with high accuracy. Besides its DNA editing ability, further-developed Cas variants can also edit the epigenome, rendering the CRISPR-Cas system a versatile tool for genome and epigenome manipulation and a pioneering force in precision medicine. This review explores the latest advancements in CRISPR-Cas technology and its therapeutic and biomedical applications, highlighting its transformative impact on precision medicine. Moreover, the current status of CRISPR therapeutics in clinical trials is discussed. Finally, we address the persisting challenges and prospects of CRISPR-Cas technology.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"6 ","pages":"1509924"},"PeriodicalIF":4.9,"publicationDate":"2024-12-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11669675/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142900999","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":"Comparison of genotyping assays for detection of targeted CRISPR/Cas mutagenesis in highly polyploid sugarcane.","authors":"Eleanor J Brant, David May, Ayman Eid, Fredy Altpeter","doi":"10.3389/fgeed.2024.1505844","DOIUrl":"10.3389/fgeed.2024.1505844","url":null,"abstract":"<p><p>Sugarcane (<i>Saccharum</i> spp.) is an important biofuel feedstock and a leading source of global table sugar. <i>Saccharum</i> hybrid cultivars are highly polyploid (2n = 100-130), containing large numbers of functionally redundant hom(e)ologs in their genomes. Genome editing with sequence-specific nucleases holds tremendous promise for sugarcane breeding. However, identification of plants with the desired level of co-editing within a pool of primary transformants can be difficult. While DNA sequencing provides direct evidence of targeted mutagenesis, it is cost-prohibitive as a primary screening method in sugarcane and most other methods of identifying mutant lines have not been optimized for use in highly polyploid species. In this study, non-sequencing methods of mutant screening, including capillary electrophoresis (CE), Cas9 RNP assay, and high-resolution melt analysis (HRMA), were compared to assess their potential for CRISPR/Cas9-mediated mutant screening in sugarcane. These assays were used to analyze sugarcane lines containing mutations at one or more of six sgRNA target sites. All three methods distinguished edited lines from wild type, with co-mutation frequencies ranging from 2% to 100%. Cas9 RNP assays were able to identify mutant sugarcane lines with as low as 3.2% co-mutation frequency, and samples could be scored based on undigested band intensity. CE was highlighted as the most comprehensive assay, delivering precise information on both mutagenesis frequency and indel size to a 1 bp resolution across all six targets. This represents an economical and comprehensive alternative to sequencing-based genotyping methods which could be applied in other polyploid species.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"6 ","pages":"1505844"},"PeriodicalIF":4.9,"publicationDate":"2024-12-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11669508/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142901002","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":"Crop genome editing through tissue-culture-independent transformation methods.","authors":"Alejandro Sebiani-Calvo, Alejandro Hernández-Soto, Götz Hensel, Andrés Gatica-Arias","doi":"10.3389/fgeed.2024.1490295","DOIUrl":"10.3389/fgeed.2024.1490295","url":null,"abstract":"<p><p>Genome editing and plant transformation are crucial techniques in plant biotechnology, allowing for the precise modification of plant genomes to enhance agronomically essential traits. The advancement of CRISPR-based genome editing tools in plants is limited, among others, by developing novel <i>in vitro</i> tissue culture methodologies for efficient plant genetic transformation. <i>In-planta</i> methodologies offer a promising alternative to overcome tissue culture limitations and facilitate crops' genetic improvement. The <i>in-planta</i> transformation methods can be categorized under the definition of means of plant genetic transformation with no or minimal tissue culture steps meeting the conditions for minimal steps: short duration with a limited number of transfers, high technical simplicity, limited list of hormones, and that the regeneration does not undergo callus development. In this review, we analyzed over 250 articles. We identified studies that follow an <i>in-planta</i> transformation methodology for delivering CRISPR/Cas9 components focusing on crop plants, as model species have been previously reviewed in detail. This approach has been successfully applied for genome editing in crop plants: camelina, cotton, lemon, melon, orange, peanut, rice, soybean, and wheat. Overall, this study underscores the importance of <i>in-planta</i> methodologies in overcoming the limitations of tissue culture and advancing the field of plant genome editing.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"6 ","pages":"1490295"},"PeriodicalIF":4.9,"publicationDate":"2024-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11655202/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142866750","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":"Genetic and physiological characteristics of <i>CsNPR3</i> edited citrus and their impact on HLB tolerance.","authors":"Trishna Tiwari, Cecile Robertson, Choaa El-Mohtar, Jude Grosser, Tripti Vashisth, Zhonglin Mou, Manjul Dutt","doi":"10.3389/fgeed.2024.1485529","DOIUrl":"10.3389/fgeed.2024.1485529","url":null,"abstract":"<p><p>Huanglongbing (HLB) disease, caused by <i>Candidatus</i> Liberibacte<i>r</i> asiaticus (<i>Ca</i>Las), severely impacts citrus production, and currently, there is no cure. Developing HLB-resistant or tolerant cultivars is crucial, with modifying defense-related genes being a promising approach to managing HLB. NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) is a positive regulator of systemic acquired resistance (SAR), which enhances resistance to pathogens, whereas NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 3 (NPR3) is a negative regulator of SAR. To unambiguously address the role of <i>CsNPR3</i> in HLB, we introduced mutations into the <i>CsNPR3</i> gene in sweet orange (<i>Citrus sinensis</i> L. Osbeck) through genome editing and assessed their effects on morphology, physiology, and resistance/tolerance to HLB. Several genome-edited 'Hamlin' sweet orange trees harboring frameshift-inducing insertions or deletions were identified. After confirming the genome editing using Sanger sequencing, selected lines were grafted onto C-146 trifoliate hybrid rootstocks for clonal propagation. The progenies were then infected with <i>Ca</i>Las using a no-choice Asian Citrus Psyllid (ACP) feeding assay. Evaluation of the genetic and physiological characteristics of <i>CsNPR3</i>-edited citrus trees under greenhouse conditions revealed that the edited trees exhibited greater vigor than the wild-type trees, despite the lack of significant differences in <i>Ca</i>Las titers. Although further field evaluation is needed, our findings indicate that <i>CsNPR3</i> contributes to HLB-caused tree deterioration and demonstrate that editing <i>CsNPR3</i> can enhance tolerance to HLB.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"6 ","pages":"1485529"},"PeriodicalIF":4.9,"publicationDate":"2024-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11652141/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142857089","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":"Towards functional maps of non-coding variants in cancer.","authors":"Yihan Wang, Gary C Hon","doi":"10.3389/fgeed.2024.1481443","DOIUrl":"10.3389/fgeed.2024.1481443","url":null,"abstract":"<p><p>Large scale cancer genomic studies in patients have unveiled millions of non-coding variants. While a handful have been shown to drive cancer development, the vast majority have unknown function. This review describes the challenges of functionally annotating non-coding cancer variants and understanding how they contribute to cancer. We summarize recently developed high-throughput technologies to address these challenges. Finally, we outline future prospects for non-coding cancer genetics to help catalyze personalized cancer therapy.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"6 ","pages":"1481443"},"PeriodicalIF":4.9,"publicationDate":"2024-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11560456/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142633510","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":"Beyond the traditional distinctions of genome editing: evaluating a vulnerability framework.","authors":"Ioanna Angelioudaki, Ana Ruxandra Badea, Martina Bodo, Daniel Fernández-Soto, Emmanouela Sevasti Karyampa, Adam Kokkinakis, Nikolaos Kolisis, Xenia Kominea, Sandra Ozáez Armijos, Simon Vogel, Oliver Feeney","doi":"10.3389/fgeed.2024.1426228","DOIUrl":"https://doi.org/10.3389/fgeed.2024.1426228","url":null,"abstract":"<p><p>Over 40 years ago, the 1982 Splicing Life report outlined the two distinctions that have orientated much of the normative and legal landscape of genetic intervention or genome editing since - that of somatic versus germline (or heritable interventions) and medical versus non-medical (or enhancement) applications. During this time, these distinctions have been used to ethically prioritize some areas of research and potential application, such as somatic treatments, while considering others for prohibition, such as germline enhancements. Nevertheless, somatic interventions may also be done for controversial enhancement purposes while some germline interventions may be done with greater <i>prima facie</i> justification (e.g., the enhancement of athletic ability versus the avoidance of Tay-Sachs disease). Even with new somatic treatments that are generally lauded, exemplified with the case of Casgevy, many issues still arise - such as cost and access, particularly salient on a global level. The concerns over a dystopian future of genetic haves and have nots, as a result of enhancement and/or germline interventions, that perhaps may happen, should not distract us from a greater attention to what is happening in the here and now. In this paper, we will highlight the limits of the two distinctions in terms of moving from questions of \"should a technology be used\" to \"how should a technology be used.\" We argue that an additional focus on vulnerability and marginalization can be useful to support the attempt to better prioritize which interventions should be permitted or prohibited. We show how this can better dovetail with calls for effective (global) governance and reasonable consensus by focusing on the most urgent issues and developing policy accordingly, while leaving aside more abstract issues for further discussion.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"6 ","pages":"1426228"},"PeriodicalIF":4.9,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11556113/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142633495","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":"Knockout mutation in <i>TaD27</i> enhances number of productive tillers in hexaploid wheat.","authors":"Muhammad Jawad Akbar Awan, Imran Amin, Awais Rasheed, Nasir A Saeed, Shahid Mansoor","doi":"10.3389/fgeed.2024.1455761","DOIUrl":"10.3389/fgeed.2024.1455761","url":null,"abstract":"<p><p>Recent advances allow the deployment of cluster regularly interspaced short palindromic repeats (CRISPR)-associated endonucleases (Cas) system for the targeted mutagenesis in the genome with accuracy and precision for trait improvement in crops. CRISPR-Cas systems have been extensively utilized to induce knockout or frameshift mutations in the targeted sequence of mostly negative regulating genes for wheat improvement. However, most of the reported work has been done in non-commercial varieties of wheat and introgression of edited alleles into breeding population comes with the penalty of unwanted linkage-drag. Wheat yield is controlled by various genes such as positive and negative regulators. The <i>TaD27</i> gene is described as a negative regulator of shoot branching or tillering and involved in the biosynthesis of strigolactones. In this study, we developed <i>Tad27</i> knockout mutant lines of an elite wheat cultivar that showed a twofold increase in the number of tillers and 1.8-fold increase in the number of grains per plant. Subsequently, enhancing the grain yield without any morphological penalty in the architecture of the plants. The co-transformation of regeneration enhancing growth regulator, G<i>rowth Regulating Factor 4</i> (<i>GRF4</i>) and its cofactor <i>GRF-Interacting Factor 1</i> (<i>GIF1</i>), under single T-DNA cassette improved the regeneration efficiency up to 6% of transgenic events from mature embryos of wheat. Our results indicate that the CRISPR-mediated targeted mutagenesis confers the potential to knockout yield-related negative regulators in elite cultivars of wheat that can substantially enhance grain yield per plant and this strategy can be harnessed for the improvement of future wheat.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"6 ","pages":"1455761"},"PeriodicalIF":4.9,"publicationDate":"2024-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11513295/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142523774","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":"Targeting DLBCL by mutation-specific disruption of cancer-driving oncogenes.","authors":"Najmeh Heshmatpour, S Maryam Kazemi, Niklas D Schmidt, Sarita R Patnaik, Patrick Korus, Bodo G C Wilkens, Arturo Macarrón Palacios","doi":"10.3389/fgeed.2024.1427322","DOIUrl":"10.3389/fgeed.2024.1427322","url":null,"abstract":"<p><p>Diffuse large B cell lymphomas (DLBCL) are highly aggressive tumors. Their genetic complexity and heterogeneity have hampered the development of novel approaches for precision medicine. Our study aimed to develop a personalized therapy for DLBCL by utilizing the CRISPR/Cas system to induce knockouts (KO) of driver genes, thereby causing cancer cell death while minimizing side effects. We focused on OCI-LY3 cells, modeling DLBCL, and compared them with BJAB cells as controls. Analysis of whole exome sequencing revealed significant mutations in genes like <i>PAX5</i>, <i>CD79B</i>, and <i>MYC</i> in OCI-LY3 cells. CRISPR/Cas9-mediated KO of these genes resulted in reduced cancer cell viability. Subsequent single and dual gRNA targeting of <i>PAX5</i> mutations inhibited proliferation specifically in OCI-LY3 cells. Moreover, dual gRNA targeting of <i>PAX5</i> and <i>MYC</i> induced chromosomal rearrangements, reducing cell proliferation substantially. However, targeting single intronic mutations did not affect cell viability, highlighting the importance of disrupting protein function. Targeting multiple mutations simultaneously addresses intra-tumoral heterogeneity, and the transient delivery of CRISPR/Cas9 allows for permanent gene disruption. While challenges such as incomplete editing efficiency and delivery limitations exist, further optimization may enhance therapeutic efficacy. Overall, our findings demonstrate the efficacy of CRISPR/Cas9 in targeting oncogenic mutations, opening avenues for precision medicine in DLBCL treatment.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"6 ","pages":"1427322"},"PeriodicalIF":4.9,"publicationDate":"2024-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11513324/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142523775","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 potential of HBV cure: an overview of CRISPR-mediated HBV gene disruption.","authors":"Zhi Q Yao, Madison B Schank, Juan Zhao, Mohamed El Gazzar, Ling Wang, Yi Zhang, Addison C Hill, Puja Banik, Jaeden S Pyburn, Jonathan P Moorman","doi":"10.3389/fgeed.2024.1467449","DOIUrl":"10.3389/fgeed.2024.1467449","url":null,"abstract":"<p><p>Hepatitis B virus (HBV) infection is a common cause of liver disease worldwide. The current antiviral treatment using nucleotide analogues (NAs) can only suppress <i>de novo</i> HBV replication but cannot eliminate chronic HBV infection due to the persistence of covalently closed circular (ccc) DNA that sustains viral replication. The CRISPR/Cas9 system is a novel genome-editing tool that enables precise gene disruption and inactivation. With high efficiency and simplicity, the CRISPR/Cas9 system has been utilized in multiple studies to disrupt the HBV genome specifically, eliciting varying anti-HBV effects both <i>in vitro</i> and <i>in vivo</i>. Additionally, multi-locus gene targeting has shown enhanced antiviral activity, paving the way for combination therapy to disrupt and inactivate HBV cccDNA as well as integrated HBV DNA. Despite its promising antiviral effects, this technology faces several challenges that need to be overcome before its clinical application, i.e., off-target effects and <i>in vivo</i> drug delivery. As such, there is a need for improvement in CRISPR/Cas9 efficiency, specificity, versatility, and delivery. Here, we critically review the recent literature describing the tools employed in designing guide RNAs (gRNAs) targeting HBV genomes, the vehicles used for expressing and delivering CRISPR/Cas9 components, the models used for evaluating CRISPR-mediated HBV gene disruption, the methods used for assessing antiviral and off-target effects induced by CRISPR/Cas9-mediated HBV gene disruption, and the prospects of future directions and challenges in leveraging this HBV gene-editing approach, to advance the HBV treatment toward a clinical cure.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"6 ","pages":"1467449"},"PeriodicalIF":4.9,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11496132/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142514123","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}