Frontiers in genome editing最新文献

{"title":"Deciphering and targeting host factors to counteract SARS-CoV-2 and coronavirus infections: insights from CRISPR approaches.","authors":"Zhifen Cui,&nbsp;Hongyan Wang,&nbsp;Yizhou Dong,&nbsp;Shan-Lu Liu,&nbsp;Qianben Wang","doi":"10.3389/fgeed.2023.1231656","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1231656","url":null,"abstract":"<p><p>Severe respiratory syndrome coronavirus 2 (SARS-CoV-2) and other coronaviruses depend on host factors for the process of viral infection and replication. A better understanding of the dynamic interplay between viral pathogens and host cells, as well as identifying of virus-host dependencies, offers valuable insights into disease mechanisms and informs the development of effective therapeutic strategies against viral infections. This review delves into the key host factors that facilitate or hinder SARS-CoV-2 infection and replication, as identified by CRISPR/Cas9-based screening platforms. Furthermore, we explore CRISPR/Cas13-based gene therapy strategies aimed at targeting these host factors to inhibit viral infection, with the ultimate goal of eradicating SARS-CoV-2 and preventing and treating related coronaviruses for future outbreaks.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1231656"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10372414/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9906744","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 library screening to develop HEK293-derived cell lines with improved lentiviral vector titers.","authors":"Brian J Iaffaldano,&nbsp;Michael P Marino,&nbsp;Jakob Reiser","doi":"10.3389/fgeed.2023.1218328","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1218328","url":null,"abstract":"<p><p>Lentiviral (LV) vectors have emerged as powerful tools for treating genetic and acquired human diseases. As clinical studies and commercial demands have progressed, there has been a growing need for large amounts of purified LV vectors. To help meet this demand, we developed CRISPR library screening methods to identify genetic perturbations in human embryonic kidney 293 (HEK293) cells and their derivatives that may increase LV vector titers. Briefly, LV vector-based Human CRISPR Activation and Knockout libraries (Calabrese and Brunello) were used to modify HEK293 and HEK293T cells. These cell populations were then expanded, and integrated LV vector genomes were rescued by transfection. LV vectors were harvested, and the process of sequential transduction and rescue-transfection was iterated. Through this workflow, guide RNAs (gRNAs) that target genes that may suppress or enhance LV vector production were enriched and identified with Next-Generation Sequencing (NGS). Though more work is needed to test genes identified in this screen, we expect that perturbations of genes we identified here, such as <i>TTLL12</i>, which is an inhibitor of antiviral innate immunity may be introduced and multiplexed to yield cell lines with improved LV vector productivity.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1218328"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10373892/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9916116","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":"Combined approaches for increasing fetal hemoglobin (HbF) and <i>de novo</i> production of adult hemoglobin (HbA) in erythroid cells from β-thalassemia patients: treatment with HbF inducers and CRISPR-Cas9 based genome editing.","authors":"Alessia Finotti,&nbsp;Roberto Gambari","doi":"10.3389/fgeed.2023.1204536","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1204536","url":null,"abstract":"<p><p>Genome editing (GE) is one of the most efficient and useful molecular approaches to correct the effects of gene mutations in hereditary monogenetic diseases, including β-thalassemia. CRISPR-Cas9 gene editing has been proposed for effective correction of the β-thalassemia mutation, obtaining high-level \"<i>de novo</i>\" production of adult hemoglobin (HbA). In addition to the correction of the primary gene mutations causing β-thalassemia, several reports demonstrate that gene editing can be employed to increase fetal hemoglobin (HbF), obtaining important clinical benefits in treated β-thalassemia patients. This important objective can be achieved through CRISPR-Cas9 disruption of genes encoding transcriptional repressors of γ-globin gene expression (such as <i>BCL11A, SOX6, KLF-1</i>) or their binding sites in the HBG promoter, mimicking non-deletional and deletional HPFH mutations. These two approaches (β-globin gene correction and genome editing of the genes encoding repressors of γ-globin gene transcription) can be, at least in theory, combined. However, since multiplex CRISPR-Cas9 gene editing is associated with documented evidence concerning possible genotoxicity, this review is focused on the possibility to combine pharmacologically-mediated HbF induction protocols with the \"<i>de novo</i>\" production of HbA using CRISPR-Cas9 gene editing.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1204536"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10387548/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9922897","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Corrigendum: Establishment of an efficient protoplast regeneration and transfection protocol for field cress (<i>Lepidium campestre</i>).","authors":"Sjur Sandgrind,&nbsp;Xueyuan Li,&nbsp;Emelie Ivarson,&nbsp;Annelie Ahlman,&nbsp;Li-Hua Zhu","doi":"10.3389/fgeed.2023.1183791","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1183791","url":null,"abstract":"<p><p>[This corrects the article DOI: 10.3389/fgeed.2021.757540.].</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1183791"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10083697/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9289493","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: CRISPR and beyond: Cutting-edge technologies for gene correction in therapeutic applications.","authors":"Ayal Hendel,&nbsp;Rasmus O Bak","doi":"10.3389/fgeed.2023.1203864","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1203864","url":null,"abstract":"Gene editing promises the ultimate cure for genetic diseases by directly correcting disease-causing variants. However, the first clinical trials have chased the “low hanging fruit” using editing strategies that rely on gene disruption by introducing double-strand DNA breaks that lead to insertions and deletions (indels) by the NHEJ pathway. Since NHEJ is constitutively active throughout the cell cycle and the default DNA repair pathway, this is by far the most efficient type of conventional gene editing as opposed to homology-directed repair (HDR). HDR relies on delivery of an exogenous repair template and this pathway is active only in the S and G2 phases of the cell cycle. These two parameters constitute challenges in clinical use of HDR since exogenous DNA is toxic in most therapeutically relevant cell types and since the inherent competition between NHEJ and HDR can be a bottleneck. However, HDR benefits from enabling precise edits to be made to the genome, thereby representing true gene editing with control over the outcome. Still, in both these modalities the DNA breaks are considered a potential source of genotoxicity due to the possibility of off-target edits and chromosomal aberrations such as translocations and chromothripsis. Next-generation gene editing tools like Base and Prime Editing that rely on DNA single strand nicking reduce the risk of such harmful events but are still limited in the scope of the edits they can generate (Anzalone et al., 2020). The newest types of editors based on CRISPR-associated transposases or CRISPR-directed integrases facilitate larger edits but are still under development and immature for clinical implementation (Yarnall et al., 2022; Tou et al., 2023). This rapidly developing toolbox is expected to broaden the application of CRISPR-based tools and other site-specific engineered nucleases to cure human disease. However, on this venture of realizing precise gene correction there are several unanswered questions and challenges to overcome, some of which we hope to address with this Research Topic on Therapeutic Gene Correction Strategies Based on CRISPR Systems or Other Engineered Site-specific Nucleases. This Research Topic covers a selection of contributions including significant scientific advances in precise genetic engineering as well as expert perspectives on recent advances. OPEN ACCESS","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"5 ","pages":"1203864"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10157280/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9431310","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":"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":"5 ","pages":"1196697"},"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}

{"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}