Frontiers in genome editing最新文献

{"title":"Targeted modification of <i>CmACO1</i> by CRISPR/Cas9 extends the shelf-life of <i>Cucumis melo</i> var. <i>reticulatus</i> melon.","authors":"Satoko Nonaka,&nbsp;Maki Ito,&nbsp;Hiroshi Ezura","doi":"10.3389/fgeed.2023.1176125","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1176125","url":null,"abstract":"<p><p>The gaseous plant hormone ethylene is a regulator of fruit shelf-life, one of the essential traits in fruits. Extending fruit shelf-life reduces food loss, thereby expected to contribute to food security. The enzyme 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) is the final step of the ethylene production pathway. Its suppression via antisense technology has been demonstrated to extend the shelf-life of melon, apple, and papaya. Genome editing technology is an innovative technique for plant breeding. Because the genome editing technology would not leave the exogenous genes in the final crop products, the crops via genome editing can be considered non-genetically modified yields; compared to conventional breeding, such as mutation breeding, the breeding term would be expected to be relatively short. These points include the advantage of this technique in utilization for commercial applications. We attempted to extend the shelf-life of the Japanese luxury melon (<i>Cucumis melo</i> var. reticulatus, 'Harukei-3') via modification of the ethylene synthesis pathway with the genome editing technology, CRISPR/Cas9 system. The Melonet-DB (https://melonet-db.dna.affrc.go.jp/ap/top) showed that the melon genome had the five <i>CmACOs</i> and the gene <i>CmACO1</i> predominantly expressed in harvested fruits. From this information, <i>CmACO1</i> was expected to be a key gene for shelf-life in melons. Based on this information, the <i>CmACO1</i> was selected as the target of the CRISPR/Cas9 system and introduced the mutation. The final product of this melon did not have any exogenous genes. The mutation was inherited for at least two generations. In the T₂ generation, the fruit phenotypes 14 days after harvest were as follows: ethylene production was reduced to one-tenth that of the wild type, pericarp colour remained green, and higher fruit firmness. Early fermentation of the fresh fruit was observed in the wild-type fruit but not in the mutant. These results show that <i>CmACO1</i> knockout via CRISPR/Cas9 extended the melon's shelf-life. Moreover, our results suggest that genome editing technology would reduce food loss and contribute to food security.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1176125"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10249633/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9673654","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":"Contamination of imported kernels by unapproved genome-edited varieties poses a major challenge for monitoring and traceability during transport and handling on a global scale: inferences from a study on feral oilseed rape in Austria.","authors":"Kathrin Pascher,&nbsp;Christa Hainz-Renetzeder,&nbsp;Michaela Jagersberger,&nbsp;Katharina Kneissl,&nbsp;Günter Gollmann,&nbsp;Gerald M Schneeweiss","doi":"10.3389/fgeed.2023.1176290","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1176290","url":null,"abstract":"<p><p>Novel techniques such as CRISPR/Cas are increasingly being applied for the development of modern crops. However, the regulatory framework for production, labelling and handling of genome-edited organisms varies worldwide. Currently, the European Commission is raising the question whether genome-edited organisms should still be regulated as genetically modified organisms in the future or whether a deregulation should be implemented. In our paper, based on the outcome of a 2-year case study on oilseed rape in Austria, we show that seed spillage during import and subsequent transport and handling activities is a key factor for the unintended dispersal of seeds into the environment, the subsequent emergence of feral oilseed rape populations and their establishment and long-term persistence in natural habitats. These facts must likewise be considered in case of genome-edited oilseed rape contaminants that might be accidentally introduced with conventional kernels. We provide evidence that in Austria a high diversity of oilseed rape genotypes, including some with alleles not known from cultivated oilseed rape in Austria, exists at sites with high seed spillage and low weed management, rendering these sites of primary concern with respect to possible escape of genome-edited oilseed rape varieties into the environment. Since appropriate detection methods for single genome-edited oilseed rape events have only recently started to be successfully developed and the adverse effects of these artificial punctate DNA exchanges remain largely unknown, tracing the transmission and spread of these genetic modifications places high requirements on their monitoring, identification, and traceability.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1176290"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10156978/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9431309","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":"Prime editing in hematopoietic stem cells-From <i>ex vivo</i> to <i>in vivo</i> CRISPR-based treatment of blood disorders.","authors":"Jonas Holst Wolff,&nbsp;Jacob Giehm Mikkelsen","doi":"10.3389/fgeed.2023.1148650","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1148650","url":null,"abstract":"<p><p>Prime editing of human hematopoietic stem cells has the potential to become a safe and efficient way of treating diseases of the blood directly in patients. By allowing site-targeted gene intervention without homology-directed repair donor templates and DNA double-stranded breaks, the invention of prime editing fuels the exploration of alternatives to conventional recombination-based <i>ex vivo</i> genome editing of hematopoietic stem cells. Prime editing is as close as we get today to a true genome editing drug that does not require a separate DNA donor. However, to adapt the technology to perform <i>in vivo</i> gene correction, key challenges remain to be solved, such as identifying effective prime editing guide RNAs for clinical targets as well as developing efficient vehicles to deliver prime editors to stem cells <i>in vivo</i>. In this review, we summarize the current progress in delivery of prime editors both <i>in vitro</i> and <i>in vivo</i> and discuss future challenges that need to be adressed to allow <i>in vivo</i> prime editing as a cure for blood disorders.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1148650"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10036844/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9197841","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-generated mouse model with humanizing single-base substitution in the <i>Gnao1</i> for safety studies of RNA therapeutics.","authors":"Anna V Polikarpova,&nbsp;Tatiana V Egorova,&nbsp;Evgenii A Lunev,&nbsp;Alexandra A Tsitrina,&nbsp;Svetlana G Vassilieva,&nbsp;Irina M Savchenko,&nbsp;Yuliya Y Silaeva,&nbsp;Alexey V Deykin,&nbsp;Maryana V Bardina","doi":"10.3389/fgeed.2023.1034720","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1034720","url":null,"abstract":"<p><p>The development of personalized medicine for genetic diseases requires preclinical testing in the appropriate animal models. GNAO1 encephalopathy is a severe neurodevelopmental disorder caused by heterozygous <i>de novo</i> mutations in the <i>GNAO1</i> gene. <i>GNAO1</i> c.607 G>A is one of the most common pathogenic variants, and the mutant protein Gαo-G203R likely adversely affects neuronal signaling. As an innovative approach, sequence-specific RNA-based therapeutics such as antisense oligonucleotides or effectors of RNA interference are potentially applicable for selective suppression of the mutant <i>GNAO1</i> transcript. While <i>in vitro</i> validation can be performed in patient-derived cells, a humanized mouse model to rule out the safety of RNA therapeutics is currently lacking. In the present work, we employed CRISPR/Cas9 technology to introduce a single-base substitution into exon 6 of the <i>Gnao1</i> to replace the murine Gly203-coding triplet (GGG) with the codon used in the human gene (GGA). We verified that genome-editing did not interfere with the Gnao1 mRNA or Gαo protein synthesis and did not alter localization of the protein in the brain structures. The analysis of blastocysts revealed the off-target activity of the CRISPR/Cas9 complexes; however, no modifications of the predicted off-target sites were detected in the founder mouse. Histological staining confirmed the absence of abnormal changes in the brain of genome-edited mice. The created mouse model with the \"humanized\" fragment of the endogenous <i>Gnao1</i> is suitable to rule out unintended targeting of the wild-type allele by RNA therapeutics directed at lowering <i>GNAO1</i> c.607 G>A transcripts.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1034720"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10106585/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9378172","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":"EU regulation of gene-edited plants-A reform proposal.","authors":"Brigitte Voigt","doi":"10.3389/fgeed.2023.1119442","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1119442","url":null,"abstract":"<p><p>This article presents a proposal on how the European Union's regulatory framework on genetically modified (GM) plants should be reformed in light of recent developments in genomic plant breeding techniques. The reform involves a three-tier system reflecting the genetic changes and resulting traits of GM plants. The article is intended to contribute to the ongoing debate over how best to regulate plant gene editing techniques in the EU.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1119442"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9972293/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9388592","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":"High-throughput sgRNA testing reveals rules for Cas9 specificity and DNA repair in tomato cells.","authors":"Ellen Slaman,&nbsp;Michiel Lammers,&nbsp;Gerco C Angenent,&nbsp;Ruud A de Maagd","doi":"10.3389/fgeed.2023.1196763","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1196763","url":null,"abstract":"<p><p>CRISPR/Cas9 technology has the potential to significantly enhance plant breeding. To determine the specificity and the mutagenic spectrum of SpCas9 in tomato, we designed 89 g(uide) RNAs targeting genes of the tomato MYB transcription factor family with varying predicted specificities. Plasmids encoding sgRNAs and Cas9 were introduced into tomato protoplasts, and target sites as well as 224 predicted off-target sites were screened for the occurrence of mutations using amplicon sequencing. Algorithms for the prediction of efficacy of the sgRNAs had little predictive power in this system. The analysis of mutations suggested predictable identity of single base insertions. Off-target mutations were found for 13 out of 89 sgRNAs and only occurred at positions with one or two mismatches (at 14 and 3 sites, respectively). We found that PAM-proximal mismatches do not preclude low frequency off-target mutations. Off-target mutations were not found at all 138 positions that had three or four mismatches. We compared off-target mutation frequencies obtained with plasmid encoding sgRNAs and Cas9 with those induced by ribonucleoprotein (RNP) transfections. The use of RNPs led to a significant decrease in relative off-target frequencies at 6 out of 17, no significant difference at 9, and an increase at 2 sites. Additionally, we show that off-target sequences with insertions or deletions relative to the sgRNA may be mutated, and should be considered during sgRNA design. Altogether, our data help sgRNA design by providing insight into the Cas9-induced double-strand break repair outcomes and the occurrence of off-target mutations.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1196763"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10279869/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10087899","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":"Editorial: Overcoming genome editing challenges in plants: new tools and nanotechnologies.","authors":"Sachin Rustgi,&nbsp;Huan Zhang,&nbsp;Tufan Mehmet Oz","doi":"10.3389/fgeed.2023.1230424","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1230424","url":null,"abstract":"Genome editing has been an active research area for the last 2 decades (Carroll, 2021). As a result, we have witnessed many breakthroughs, from the development of designer nucleases to their use in microbes, animals, humans, and agricultural plants (Adli, 2018; Zhang et al., 2019; Anzalone et al., 2020; Li et al., 2023; Wang and Doudna, 2023). More recently, to improve the editing accuracy and precision and reduce dependence on the cell’s developmental state, new approaches, such as the OMEGA (obligate mobile element-guided activity), CAST (CRISPR-Cas-associated transposon), and INTEGRATE (insertion of transposable elements by guide RNA-assisted targeting), were developed and tested in different organisms (Tenjo-Castaño et al., 2022). Further, CRISPR technology was deployed in imaging, diagnostics (Wang and Doudna, 2023), and treatment of major human disorders (Wang and Doudna, 2023). Likewise, these technologies were deployed in agriculture to edit all major row crops, such as rice, wheat, maize, cotton, soybean, and horticultural crops like banana, tomato, apple, and poplar, for various traits from disease resistance to consumer preference traits (Zhu et al., 2020; FAO, 2023). Despite the revolutionary nature of genome-editing tools and the notable progress that these tools have enabled in plant improvement, there remain many challenges for the mainstream application of CRISPR technology in many plant species. Most of these challenges stem directly or indirectly from the cargo delivery and tissue culture-based plant regeneration bottlenecks (Rustgi et al.). Recent progress has been made in the delivery area through using nanomaterials and DNA/RNA viruses, along with notable improvements to the tissue culture process via developmental regulators, growth factors, and haploidy inducers, among many other approaches (Rustgi et al.). Given the remarkable amount of research on genome editing, there are still some bottlenecks, making it imperative to summarize the progress and identify areas that need further research. Keeping this goal in mind, we invited research and review papers from the leading research group in this workspace. After the extensive peer-review process, five articles that summarize the depth of the subject area were published. The articles published in this volume are briefly summarized below. OPEN ACCESS","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1230424"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10267455/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9660024","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":"Editorial: Genome and transcriptome editing to understand and treat neuromuscular diseases.","authors":"Rika Maruyama,&nbsp;Alyson Fiorillo,&nbsp;Christopher Heier,&nbsp;Dongsheng Duan,&nbsp;Toshifumi Yokota","doi":"10.3389/fgeed.2023.1176699","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1176699","url":null,"abstract":"Neuromuscular diseases such as Duchenne muscular dystrophy and facioscapulohumeral muscular dystrophy are debilitating conditions that affect millions of individuals worldwide. In recent years, there has been a growing interest in the use of genome and transcriptome editing techniques to understand and treat these diseases. This Research Topic brings together four articles that highlight the latest advances in this field. The first article “A Single Transcript Knockdown-Replacement Strategy Employing 5′ UTR Secondary Structures to Precisely Titrate Rescue Protein Translation” by Millette et al., presents a new strategy for precisely titrating rescue protein translation in cases of diseases caused by coding mutations such as amyotrophic lateral sclerosis (ALS). The authors developed amodular, single-transgene expression system that allows control over translation from high-expression, ubiquitous promoters. This system uses “attenuator” sequences in the 5’UTRwhich predictably diminish the translation of the paired gene, providing wide general utility. The authors also demonstrate that this approach can be used to achieve a knockdown and rescue effect by pairing microRNA-adapted shRNAs alongside their respective replacement gene on a single transcript. They also showed that this approach can be used to replace the SOD1 gene in stable cell lines and demonstrate complete and predictable control over replacement of SOD1 by varying the strength of attenuators. This study highlights the potential utility of this approach in treating monogenic diseases caused by heterogeneous mutations. The second article “Development of Therapeutic RNA Manipulation for Muscular Dystrophy” by Saifullah et al., reviews the current state of therapeutic RNA manipulation for muscular dystrophies, specifically Duchenne muscular dystrophy (DMD). DMD is a severe monogenic disease caused by mutations in the DMD gene, leading to muscle degeneration and atrophy early in life and premature death. This article highlights the potential of oligonucleotide-based therapeutics, specifically exon skipping using antisense oligonucleotides (ASO), as a promising strategy for treating OPEN ACCESS","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1176699"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10043468/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9220629","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":"High-efficiency editing in hematopoietic stem cells and the HUDEP-2 cell line based on <i>in vitro</i> mRNA synthesis.","authors":"Nikoletta Y Papaioannou,&nbsp;Petros Patsali,&nbsp;Basma Naiisseh,&nbsp;Panayiota L Papasavva,&nbsp;Lola Koniali,&nbsp;Ryo Kurita,&nbsp;Yukio Nakamura,&nbsp;Soteroula Christou,&nbsp;Maria Sitarou,&nbsp;Claudio Mussolino,&nbsp;Toni Cathomen,&nbsp;Marina Kleanthous,&nbsp;Carsten W Lederer","doi":"10.3389/fgeed.2023.1141618","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1141618","url":null,"abstract":"<p><p><b>Introduction:</b> Genome editing tools, such as CRISPR/Cas, TALE nucleases and, more recently, double-strand-break-independent editors, have been successfully used for gene therapy and reverse genetics. Among various challenges in the field, tolerable and efficient delivery of editors to target cells and sites, as well as independence from commercially available tools for flexibility and fast adoption of new editing technology are the most pressing. For many hematopoietic research applications, primary CD34⁺ cells and the human umbilical cord-derived progenitor erythroid 2 (HUDEP-2) cell line are highly informative substrates and readily accessible for <i>in vitro</i> manipulation. Moreover, <i>ex vivo</i> editing of CD34⁺ cells has immediate therapeutic relevance. Both cell types are sensitive to standard transfection procedures and reagents, such as lipofection with plasmid DNA, calling for more suitable methodology in order to achieve high efficiency and tolerability of editing with editors of choice. These challenges can be addressed by RNA delivery, either as a mixture of guide RNA and mRNA for CRISRP/Cas-based systems or as a mixture of mRNAs for TALENs. Compared to ribonucleoproteins or proteins, RNA as vector creates flexibility by removing dependence on commercial availability or laborious in-house preparations of novel editor proteins. Compared to DNA, RNA is less toxic and by obviating nuclear transcription and export of mRNA offers faster kinetics and higher editing efficiencies. <b>Methods:</b> Here, we detail an <i>in vitro</i> transcription protocol based on plasmid DNA templates with the addition of Anti-Reverse Cap Analog (ARCA) using T7 RNA polymerase, and poly (A) tailing using poly (A) polymerase, combined with nucleofection of HUDEP-2 and patient-derived CD34⁺ cells. Our protocol for RNA-based delivery employs widely available reagents and equipment and can easily be adopted for universal <i>in vitro</i> delivery of genome editing tools. <b>Results and Discussion:</b> Drawing on a common use case, we employ the protocol to target a β-globin mutation and to reactivate γ-globin expression as two potential therapies for β-hemoglobinopathies, followed by erythroid differentiation and functional analyses. Our protocol allows high editing efficiencies and unimpaired cell viability and differentiation, with scalability, suitability for functional assessment of editing outcomes and high flexibility in the application to different editors.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1141618"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10030607/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10296309","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":"Establishment of a genome editing tool using CRISPR-Cas9 ribonucleoprotein complexes in the non-model plant pathogen <i>Sphaerulina musiva</i>.","authors":"Joanna Tannous,&nbsp;Cole Sawyer,&nbsp;Md Mahmudul Hassan,&nbsp;Jesse L Labbe,&nbsp;Carrie Eckert","doi":"10.3389/fgeed.2023.1110279","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1110279","url":null,"abstract":"<p><p>CRISPR-Cas9 is a versatile genome editing system widely used since 2013 to introduce site-specific modifications into the genomes of model and non-model species. This technology is used in various applications, from gene knock-outs, knock-ins, and over-expressions to more precise changes, such as the introduction of nucleotides at a targeted locus. CRISPR-Cas9 has been demonstrated to be easy to establish in new species and highly efficient and specific compared to previous gene editing strategies such as Zinc finger nucleases and transcription activator-like effector nucleases. Grand challenges for emerging CRISPR-Cas9 tools in filamentous fungi are developing efficient transformation methods for non-model organisms. In this paper, we have leveraged the establishment of CRISPR-Cas9 genome editing tool that relies on Cas9/sgRNA ribonucleoprotein complexes (RNPs) in the model species <i>Trichoderma reesei</i> and developed the first protocol to efficiently transform the non-model species, <i>Sphaerulina musiva.</i> This fungal pathogen constitutes a real threat to the genus <i>Populus,</i> a foundational bioenergy crop used for biofuel production. Herein, we highlight the general considerations to design sgRNAs and their computational validation. We also describe the use of isolated protoplasts to deliver the CRISPR-Cas9 RNP components in both species and the screening for targeted genome editing events. The development of engineering tools in <i>S. musiva</i> can be used for studying genes involved in diverse processes such as secondary metabolism, establishment, and pathogenicity, among many others, but also for developing genetic mitigation approaches. The approach described here provides guidance for potential development of transformation systems in other non-model spore-bearing ascomycetes.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1110279"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10401582/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10308295","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}