Frontiers in genome editing最新文献

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Similar deamination activities but different phenotypic outcomes induced by APOBEC3 enzymes in breast epithelial cells. APOBEC3酶在乳腺上皮细胞中诱导的脱氨活性相似但表型结果不同。
Frontiers in genome editing Pub Date : 2023-01-01 DOI: 10.3389/fgeed.2023.1196697
Milaid Granadillo Rodríguez, Lai Wong, Linda Chelico
{"title":"Similar deamination activities but different phenotypic outcomes induced by APOBEC3 enzymes in breast epithelial cells.","authors":"Milaid Granadillo Rodríguez,&nbsp;Lai Wong,&nbsp;Linda Chelico","doi":"10.3389/fgeed.2023.1196697","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1196697","url":null,"abstract":"<p><p>APOBEC3 (A3) enzymes deaminate cytosine to uracil in viral single-stranded DNA as a mutagenic barrier for some viruses. A3-induced deaminations can also occur in human genomes resulting in an endogenous source of somatic mutations in multiple cancers. However, the roles of each A3 are unclear since few studies have assessed these enzymes in parallel. Thus, we developed stable cell lines expressing A3A, A3B, or A3H Hap I using non-tumorigenic MCF10A and tumorigenic MCF7 breast epithelial cells to assess their mutagenic potential and cancer phenotypes in breast cells. The activity of these enzymes was characterized by γH2AX foci formation and <i>in vitro</i> deamination. Cell migration and soft agar colony formation assays assessed cellular transformation potential. We found that all three A3 enzymes had similar γH2AX foci formation, despite different deamination activities <i>in vitro</i>. Notably, in nuclear lysates, the <i>in vitro</i> deaminase activity of A3A, A3B, and A3H did not require digestion of cellular RNA, in contrast to that of A3B and A3H in whole-cell lysates. Their similar activities in cells, nonetheless, resulted in distinct phenotypes where A3A decreased colony formation in soft agar, A3B decreased colony formation in soft agar after hydroxyurea treatment, and A3H Hap I promoted cell migration. Overall, we show that <i>in vitro</i> deamination data do not always reflect cell DNA damage, all three A3s induce DNA damage, and the impact of each is different.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10267419/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9660025","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}
引用次数: 0
Targeted modification of CmACO1 by CRISPR/Cas9 extends the shelf-life of Cucumis melo var. reticulatus melon. 利用CRISPR/Cas9靶向修饰CmACO1,延长了甜瓜的贮藏期。
Frontiers in genome editing Pub Date : 2023-01-01 DOI: 10.3389/fgeed.2023.1176125
Satoko Nonaka, Maki Ito, Hiroshi Ezura
{"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 T<sub>2</sub> 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":null,"pages":null},"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}
引用次数: 1
Prime editing in hematopoietic stem cells-From ex vivo to in vivo CRISPR-based treatment of blood disorders. 造血干细胞的启动编辑——从体外到体内基于crispr的血液疾病治疗
Frontiers in genome editing Pub Date : 2023-01-01 DOI: 10.3389/fgeed.2023.1148650
Jonas Holst Wolff, Jacob Giehm Mikkelsen
{"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":null,"pages":null},"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}
引用次数: 2
CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics. CRISPR/ cas9生成的小鼠模型,人源化Gnao1单碱基替代,用于RNA治疗药物的安全性研究。
Frontiers in genome editing Pub Date : 2023-01-01 DOI: 10.3389/fgeed.2023.1034720
Anna V Polikarpova, Tatiana V Egorova, Evgenii A Lunev, Alexandra A Tsitrina, Svetlana G Vassilieva, Irina M Savchenko, Yuliya Y Silaeva, Alexey V Deykin, Maryana V Bardina
{"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":null,"pages":null},"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}
引用次数: 0
EU regulation of gene-edited plants-A reform proposal. 欧盟对基因编辑植物的监管——一项改革提案。
Frontiers in genome editing Pub Date : 2023-01-01 DOI: 10.3389/fgeed.2023.1119442
Brigitte Voigt
{"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":null,"pages":null},"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}
引用次数: 2
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. 未经批准的基因组编辑品种对进口籽粒的污染对全球范围内运输和处理过程中的监测和可追溯性构成了重大挑战:来自奥地利一项野生油菜研究的推论。
Frontiers in genome editing Pub Date : 2023-01-01 DOI: 10.3389/fgeed.2023.1176290
Kathrin Pascher, Christa Hainz-Renetzeder, Michaela Jagersberger, Katharina Kneissl, Günter Gollmann, Gerald M Schneeweiss
{"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":null,"pages":null},"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}
引用次数: 0
Editorial: Overcoming genome editing challenges in plants: new tools and nanotechnologies. 社论:克服植物基因组编辑的挑战:新工具和纳米技术。
Frontiers in genome editing Pub Date : 2023-01-01 DOI: 10.3389/fgeed.2023.1230424
Sachin Rustgi, Huan Zhang, Tufan Mehmet Oz
{"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":null,"pages":null},"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}
引用次数: 0
Editorial: Genome and transcriptome editing to understand and treat neuromuscular diseases. 编辑:基因组和转录组编辑理解和治疗神经肌肉疾病。
Frontiers in genome editing Pub Date : 2023-01-01 DOI: 10.3389/fgeed.2023.1176699
Rika Maruyama, Alyson Fiorillo, Christopher Heier, Dongsheng Duan, Toshifumi Yokota
{"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":null,"pages":null},"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}
引用次数: 0
High-throughput sgRNA testing reveals rules for Cas9 specificity and DNA repair in tomato cells. 高通量sgRNA检测揭示了番茄细胞中Cas9特异性和DNA修复的规律。
Frontiers in genome editing Pub Date : 2023-01-01 DOI: 10.3389/fgeed.2023.1196763
Ellen Slaman, Michiel Lammers, Gerco C Angenent, Ruud A de Maagd
{"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":null,"pages":null},"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}
引用次数: 1
High-efficiency editing in hematopoietic stem cells and the HUDEP-2 cell line based on in vitro mRNA synthesis. 基于体外mRNA合成的造血干细胞和HUDEP-2细胞系的高效编辑
Frontiers in genome editing Pub Date : 2023-01-01 DOI: 10.3389/fgeed.2023.1141618
Nikoletta Y Papaioannou, Petros Patsali, Basma Naiisseh, Panayiota L Papasavva, Lola Koniali, Ryo Kurita, Yukio Nakamura, Soteroula Christou, Maria Sitarou, Claudio Mussolino, Toni Cathomen, Marina Kleanthous, Carsten W Lederer
{"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<sup>+</sup> 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<sup>+</sup> 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<sup>+</sup> 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":null,"pages":null},"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}
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