Frontiers in genome editing最新文献

{"title":"Genetic manipulation of betta fish.","authors":"Alec Palmiotti, Madison R Lichak, Pei-Yin Shih, Young Mi Kwon, Andres Bendesky","doi":"10.3389/fgeed.2023.1167093","DOIUrl":"10.3389/fgeed.2023.1167093","url":null,"abstract":"<p><p><i>Betta splendens,</i> also known as Siamese fighting fish or \"betta,\" is a freshwater fish species renowned for its astonishing morphological diversity and extreme aggressive behavior. Despite recent advances in our understanding of the genetics and neurobiology of betta, the lack of tools to manipulate their genome has hindered progress at functional and mechanistic levels. In this study, we outline the use of three genetic manipulation technologies, which we have optimized for use in betta: CRISPR/Cas9-mediated knockout, CRISPR/Cas9-mediated knockin, and Tol2-mediated transgenesis. We knocked out three genes: <i>alkal2l, bco1l,</i> and <i>mitfa,</i> and analyzed their effects on viability and pigmentation. Furthermore, we knocked in a fluorescent protein into the <i>mitfa</i> locus, a proof-of-principle experiment of this powerful technology in betta. Finally, we used Tol2-mediated transgenesis to create fish with ubiquitous expression of GFP, and then developed a bicistronic plasmid with heart-specific expression of a red fluorescent protein to serve as a visible marker of successful transgenesis. Our work highlights the potential for the genetic manipulation of betta, providing valuable resources for the effective use of genetic tools in this animal model.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1167093"},"PeriodicalIF":0.0,"publicationDate":"2023-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10401044/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10325328","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":"Strategies for delivery of CRISPR/Cas-mediated genome editing to obtain edited plants directly without transgene integration.","authors":"Zuzana Kocsisova, Viktoriya Coneva","doi":"10.3389/fgeed.2023.1209586","DOIUrl":"10.3389/fgeed.2023.1209586","url":null,"abstract":"<p><p>Increased understanding of plant genetics and the development of powerful and easier-to-use gene editing tools over the past century have revolutionized humankind's ability to deliver precise genotypes in crops. Plant transformation techniques are well developed for making transgenic varieties in certain crops and model organisms, yet reagent delivery and plant regeneration remain key bottlenecks to applying the technology of gene editing to most crops. Typical plant transformation protocols to produce transgenic, genetically modified (GM) varieties rely on transgenes, chemical selection, and tissue culture. Typical protocols to make gene edited (GE) varieties also use transgenes, even though these may be undesirable in the final crop product. In some crops, the transgenes are routinely segregated away during meiosis by performing crosses, and thus only a minor concern. In other crops, particularly those propagated vegetatively, complex hybrids, or crops with long generation times, such crosses are impractical or impossible. This review highlights diverse strategies to deliver CRISPR/Cas gene editing reagents to regenerable plant cells and to recover edited plants without unwanted integration of transgenes. Some examples include delivering DNA-free gene editing reagents such as ribonucleoproteins or mRNA, relying on reagent expression from non-integrated DNA, using novel delivery mechanisms such as viruses or nanoparticles, using unconventional selection methods to avoid integration of transgenes, and/or avoiding tissue culture altogether. These methods are advancing rapidly and already enabling crop scientists to make use of the precision of CRISPR gene editing tools.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1209586"},"PeriodicalIF":4.9,"publicationDate":"2023-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10398581/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10005816","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":"Methods of crop improvement and applications towards fortifying food security.","authors":"Aayushi Patel,&nbsp;Andrew Miles,&nbsp;Tara Strackhouse,&nbsp;Logan Cook,&nbsp;Sining Leng,&nbsp;Shrina Patel,&nbsp;Kelsey Klinger,&nbsp;Sairam Rudrabhatla,&nbsp;Shobha D Potlakayala","doi":"10.3389/fgeed.2023.1171969","DOIUrl":"10.3389/fgeed.2023.1171969","url":null,"abstract":"<p><p>Agriculture has supported human life from the beginning of civilization, despite a plethora of biotic (pests, pathogens) and abiotic (drought, cold) stressors being exerted on the global food demand. In the past 50 years, the enhanced understanding of cellular and molecular mechanisms in plants has led to novel innovations in biotechnology, resulting in the introduction of desired genes/traits through plant genetic engineering. Targeted genome editing technologies such as Zinc-Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) have emerged as powerful tools for crop improvement. This new CRISPR technology is proving to be an efficient and straightforward process with low cost. It possesses applicability across most plant species, targets multiple genes, and is being used to engineer plant metabolic pathways to create resistance to pathogens and abiotic stressors. These novel genome editing (GE) technologies are poised to meet the UN's sustainable development goals of \"zero hunger\" and \"good human health and wellbeing.\" These technologies could be more efficient in developing transgenic crops and aid in speeding up the regulatory approvals and risk assessments conducted by the US Departments of Agriculture (USDA), Food and Drug Administration (FDA), and Environmental Protection Agency (EPA).</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1171969"},"PeriodicalIF":0.0,"publicationDate":"2023-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10361821/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10241066","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":"Exploring the future of GM technology in sustainable local food systems in Colombia.","authors":"Néstor Julián Cárdenas Pardo, Dolly Esperanza Rodriguez Robayo, John Cristhian Fernandez Lizarazo, Diego Camilo Peña-Quemba, Erica McGale","doi":"10.3389/fgeed.2023.1181811","DOIUrl":"10.3389/fgeed.2023.1181811","url":null,"abstract":"<p><p>The security of Earth's food systems is challenged by shifting regional climates. While agricultural processes are disrupted by climate change, they also play a large role in contributing to destabilizing greenhouse gases. Finding new strategies to increase yields while decreasing agricultural environmental impacts is essential. Tropical agriculture is particularly susceptible to climate change: local, smallholder farming, which provides a majority of the food supply, is high risk and has limited adaptation capacity. Rapid, inexpensive, intuitive solutions are needed, like the implementation of genetically modified (GM) crops. In the Latin American tropics, high awareness and acceptance of GM technologies, opportunities to test GM crops as part of local agricultural educations, and their known economic benefits, support their use. However, this is not all that is needed for the future of GM technologies in these areas: GM implementation must also consider environmental and social sustainability, which can be unique to a locality. Primarily from the perspective of its educators, the potential of a rural Colombian university in driving GM implementation is explored, including the role of this type of university in producing agricultural engineers who can innovate with GM to meet regionally-dependent environmental and cultural needs that could increase their sustainability.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1181811"},"PeriodicalIF":4.9,"publicationDate":"2023-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10349173/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10202801","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":"Assessment of anti-HIV-1 guide RNA efficacy in cells containing the viral target sequence, corresponding gRNA, and CRISPR/Cas9.","authors":"Alexander G Allen, Cheng-Han Chung, Stephen D Worrell, Glad Nwaozo, Rebekah Madrid, Anthony R Mele, Will Dampier, Michael R Nonnemacher, Brian Wigdahl","doi":"10.3389/fgeed.2023.1101483","DOIUrl":"10.3389/fgeed.2023.1101483","url":null,"abstract":"<p><p>The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing system has been shown to be effective at inhibiting human immunodeficiency virus type 1 (HIV-1). Studies have not consistently used a trackable dual reporter system to determine what cells received the Cas9/gRNA to determine the overall knockdown of HIV. Some studies have used stably transduced cells under drug selection to accomplish this goal. Here a two-color system was used that allows tracking of viral protein expression and which cells received the CRISPR/Cas9 system. These experiments ensured that each gRNA used was a perfect match to the intended target to remove this variable. The data showed that gRNAs targeting the transactivation response element (TAR) region or other highly conserved regions of the HIV-1 genome were effective at stopping viral gene expression, with multiple assays demonstrating greater than 95 percent reduction. Conversely, gRNAs targeting conserved sites of the 5' portion of the U3 region were largely ineffective, demonstrating that the location of edits in the long terminal repeat (LTR) matter with respect to function. In addition, it was observed that a gRNA targeting Tat was effective in a T-cell model of HIV-1 latency. Taken together, these studies demonstrated gRNAs designed to highly conserved functional regions have near 100% efficacy <i>in vitro</i> in cells known to have received the Cas9/gRNA pair.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1101483"},"PeriodicalIF":4.9,"publicationDate":"2023-04-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10134072/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9393688","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":"Genome editing for improving nutritional quality, post-harvest shelf life and stress tolerance of fruits, vegetables, and ornamentals.","authors":"Punam Sharma, Anuradha Pandey, Rinku Malviya, Sharmistha Dey, Subhasis Karmakar, Dipak Gayen","doi":"10.3389/fgeed.2023.1094965","DOIUrl":"10.3389/fgeed.2023.1094965","url":null,"abstract":"<p><p>Agricultural production relies on horticultural crops, including vegetables, fruits, and ornamental plants, which sustain human life. With an alarming increase in human population and the consequential need for more food, it has become necessary for increased production to maintain food security. Conventional breeding has subsidized the development of improved verities but to enhance crop production, new breeding techniques need to be acquired. CRISPR-Cas9 system is a unique and powerful genome manipulation tool that can change the DNA in a precise way. Based on the bacterial adaptive immune system, this technique uses an endonuclease that creates double-stranded breaks (DSBs) at the target loci under the guidance of a single guide RNA. These DSBs can be repaired by a cellular repair mechanism that installs small insertion and deletion (indels) at the cut sites. When equated to alternate editing tools like ZFN, TALENs, and meganucleases, CRISPR- The cas-based editing tool has quickly gained fast-forward for its simplicity, ease to use, and low off-target effect. In numerous horticultural and industrial crops, the CRISPR technology has been successfully used to enhance stress tolerance, self-life, nutritional improvements, flavor, and metabolites. The CRISPR-based tool is the most appropriate one with the prospective goal of generating non-transgenic yields and avoiding the regulatory hurdles to release the modified crops into the market. Although several challenges for editing horticultural, industrial, and ornamental crops remain, this new novel nuclease, with its crop-specific application, makes it a dynamic tool for crop improvement.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1094965"},"PeriodicalIF":4.9,"publicationDate":"2023-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9998953/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9096999","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":"T-CAST: An optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds.","authors":"Manuel Rhiel, Kerstin Geiger, Geoffroy Andrieux, Julia Rositzka, Melanie Boerries, Toni Cathomen, Tatjana I Cornu","doi":"10.3389/fgeed.2023.1130736","DOIUrl":"10.3389/fgeed.2023.1130736","url":null,"abstract":"<p><p>Transcription activator-like effector nucleases (TALENs) are programmable nucleases that have entered the clinical stage. Each subunit of the dimer consists of a DNA-binding domain composed of an array of TALE repeats fused to the catalytically active portion of the FokI endonuclease. Upon DNA-binding of both TALEN arms in close proximity, the FokI domains dimerize and induce a staggered-end DNA double strand break. In this present study, we describe the implementation and validation of TALEN-specific CAST-Seq (T-CAST), a pipeline based on CAST-Seq that identifies TALEN-mediated off-target effects, nominates off-target sites with high fidelity, and predicts the TALEN pairing conformation leading to off-target cleavage. We validated T-CAST by assessing off-target effects of two promiscuous TALENs designed to target the <i>CCR5</i> and <i>TRAC</i> loci. Expression of these TALENs caused high levels of translocations between the target sites and various off-target sites in primary T cells. Introduction of amino acid substitutions to the FokI domains, which render TALENs obligate-heterodimeric (OH-TALEN), mitigated the aforementioned off-target effects without loss of on-target activity. Our findings highlight the significance of T-CAST to assess off-target effects of TALEN designer nucleases and to evaluate mitigation strategies, and advocate the use of obligate-heterodimeric TALEN scaffolds for therapeutic genome editing.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1130736"},"PeriodicalIF":4.9,"publicationDate":"2023-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9986454/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9075311","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":"Rare immune diseases paving the road for genome editing-based precision medicine.","authors":"Mara Pavel-Dinu, Simon Borna, Rosa Bacchetta","doi":"10.3389/fgeed.2023.1114996","DOIUrl":"10.3389/fgeed.2023.1114996","url":null,"abstract":"<p><p>Clustered regularly interspaced short palindromic repeats (CRISPR) genome editing platform heralds a new era of gene therapy. Innovative treatments for life-threatening monogenic diseases of the blood and immune system are transitioning from semi-random gene addition to precise modification of defective genes. As these therapies enter first-in-human clinical trials, their long-term safety and efficacy will inform the future generation of genome editing-based medicine. Here we discuss the significance of Inborn Errors of Immunity as disease prototypes for establishing and advancing precision medicine. We will review the feasibility of clustered regularly interspaced short palindromic repeats-based genome editing platforms to modify the DNA sequence of primary cells and describe two emerging genome editing approaches to treat <i>RAG2</i> deficiency, a primary immunodeficiency, and <i>FOXP3</i> deficiency, a primary immune regulatory disorder.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1114996"},"PeriodicalIF":4.9,"publicationDate":"2023-02-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9945114/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10790737","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":"Whole genome sequencing of <i>CCR5</i> CRISPR-Cas9-edited Mauritian cynomolgus macaque blastomeres reveals large-scale deletions and off-target edits.","authors":"Jenna Kropp Schmidt, Yun Hee Kim, Nick Strelchenko, Sarah R Gierczic, Derek Pavelec, Thaddeus G Golos, Igor I Slukvin","doi":"10.3389/fgeed.2022.1031275","DOIUrl":"10.3389/fgeed.2022.1031275","url":null,"abstract":"<p><p><b>Introduction:</b> Genome editing by CRISPR-Cas9 approaches offers promise for introducing or correcting disease-associated mutations for research and clinical applications. Nonhuman primates are physiologically closer to humans than other laboratory animal models, providing ideal candidates for introducing human disease-associated mutations to develop models of human disease. The incidence of large chromosomal anomalies in CRISPR-Cas9-edited human embryos and cells warrants comprehensive genotypic investigation of editing outcomes in primate embryos. Our objective was to evaluate on- and off-target editing outcomes in <i>CCR5</i> CRISPR-Cas9-targeted Mauritian cynomolgus macaque embryos. <b>Methods:</b> DNA isolated from individual blastomeres of two embryos, along with paternal and maternal DNA, was subjected to whole genome sequencing (WGS) analysis. <b>Results:</b> Large deletions were identified in macaque blastomeres at the on-target site that were not previously detected using PCR-based methods. <i>De novo</i> mutations were also identified at predicted CRISPR-Cas9 off-target sites. <b>Discussion:</b> This is the first report of WGS analysis of CRISPR-Cas9-targeted nonhuman primate embryonic cells, in which a high editing efficiency was coupled with the incidence of editing errors in cells from two embryos. These data demonstrate that comprehensive sequencing-based methods are warranted for evaluating editing outcomes in primate embryos, as well as any resultant offspring to ensure that the observed phenotype is due to the targeted edit and not due to unidentified off-target mutations.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"4 ","pages":"1031275"},"PeriodicalIF":4.9,"publicationDate":"2023-01-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9877282/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9224435","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":"CRISPR/Cas9 mediated gene editing in non-model nematode <i>Panagrolaimus</i> sp. PS1159.","authors":"Viktoria Hellekes,&nbsp;Denise Claus,&nbsp;Johanna Seiler,&nbsp;Felix Illner,&nbsp;Philipp H Schiffer,&nbsp;Michael Kroiher","doi":"10.3389/fgeed.2023.1078359","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1078359","url":null,"abstract":"<p><p>The phylum Nematoda harbors a huge diversity of species in a broad range of ecosystems and habitats. Nematodes share a largely conserved Bauplan but major differences have been found in early developmental processes. The development of the nematode model organism <i>Caenorhabditis elegans</i> has been studied in great detail for decades. These efforts have provided the community with a large number of protocols and methods. Unfortunately, many of these tools are not easily applicable in non-<i>Caenorhabditis</i> nematodes. In recent years it has become clear that many crucial genes in the <i>C. elegans</i> developmental toolkit are absent in other nematode species. It is thus necessary to study the developmental program of other nematode species in detail to understand evolutionary conservation and novelty in the phylum. <i>Panagrolaimus</i> sp. PS1159 is a non-parasitic nematode exhibiting parthenogenetic reproduction and we are establishing the species to comparatively study evolution, biodiversity, and alternative reproduction and survival strategies. Here, we demonstrate the first successful application of the CRISPR/Cas9 system for genome editing in <i>Panagrolaimus</i> sp. PS1159 and the closely related hermaphroditic species <i>Propanagrolaimus</i> sp. JU765 applying the non-homologous end joining and the homology-directed repair (HDR) mechanisms. Using microinjections and modifying published protocols from <i>C. elegans</i> and <i>P. pacificus</i> we induced mutations in the orthologue of <i>unc-22.</i> This resulted in a visible uncoordinated twitching phenotype. We also compared the HDR efficiency following the delivery of different single-stranded oligodeoxynucleotides (ssODNs). Our work will expand the applicability for a wide range of non-model nematodes from across the tree and facilitate functional analysis into the evolution of parthenogenesis, changes in the developmental program of Nematoda, and cryptobiosis.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1078359"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9935820/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10756255","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}