Frontiers in genome editing最新文献

{"title":"What do people think about genetic engineering? A systematic review of questionnaire surveys before and after the introduction of CRISPR","authors":"Pedro Dias Ramos, Maria Strecht Almeida, Ingrid Anna Sofia Olsson","doi":"10.3389/fgeed.2023.1284547","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1284547","url":null,"abstract":"The advent of CRISPR-Cas9 in 2012 started revolutionizing the field of genetics by broadening the access to a method for precise modification of the human genome. It also brought renewed attention to the ethical issues of genetic modification and the societal acceptance of technology for this purpose. So far, many surveys assessing public attitudes toward genetic modification have been conducted worldwide. Here, we present the results of a systematic review of primary publications of surveys addressing public attitudes toward genetic modification as well as the awareness and knowledge about the technology required for genetic modification. A total of 53 primary publications (1987–2020) focusing on applications in humans and non-human animals were identified, covering countries in four continents. Of the 53 studies, 30 studies from until and including 2012 (pre-CRISPR) address gene therapy in humans and genetic modification of animals for food production and biomedical research. The remaining 23 studies from after 2013 (CRISPR) address gene editing in humans and animals. Across countries, respondents see gene therapy for disease treatment or prevention in humans as desirable and highly acceptable, whereas enhancement is generally met with opposition. When the study distinguishes between somatic and germline applications, somatic gene editing is generally accepted, whereas germline applications are met with ambivalence. The purpose of the application is also important for assessing attitudes toward genetically modified animals: modification in food production is much less accepted than for biomedical application in pre-CRISPR studies. A relationship between knowledge/awareness and attitude toward genetic modification is often present. A critical appraisal of methodology quality in the primary publications with regards to sampling and questionnaire design, development, and administration shows that there is considerable scope for improvement in the reporting of methodological detail. Lack of information is more common in earlier studies, which probably reflects the changing practice in the field.","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139172450","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Diversity of transgene integration and gene-editing events in wheat (<i>Triticum aestivum</i> L.) transgenic plants generated using <i>Agrobacterium</i>-mediated transformation.","authors":"Louie Cris Lopos, Natalia V Bykova, Janeen Robinson, Susan Brown, Kerry Ward, Andriy Bilichak","doi":"10.3389/fgeed.2023.1265103","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1265103","url":null,"abstract":"<p><p>Improvement in agronomic traits in crops through gene editing (GE) relies on efficient transformation protocols for delivering the CRISPR/Cas9-coded transgenes. Recently, a few embryogenesis-related genes have been described, the co-delivery of which significantly increases the transformation efficiency with reduced genotype-dependency. Here, we characterized the transgenic and GE events in wheat (cv. Fielder) when transformed with <i>GROWTH-REGULATING FACTOR 4</i> (<i>GRF4</i>) and its cofactor <i>GRF-INTERACTING FACTOR 1</i> (<i>GIF1</i>) chimeric gene. Transformation efficiency in our experiments ranged from 22% to 68%, and the editing events were faithfully propagated into the following generation. Both low- and high-copy-number integration events were recovered in the T₀ population with various levels of integrity of the left and right T-DNA borders. We also generated a population of wheat plants with 10 different gRNAs targeting 30 loci in the genome. A comparison of the epigenetic profiles at the target sites and editing efficiency revealed a significant positive correlation between chromatin accessibility and mutagenesis rate. Overall, the preliminary screening of transgene quality and GE events in the T₀ population of plants regenerated through the co-delivery of <i>GRF-GIF</i> can allow for the propagation of the best candidates for further phenotypic analysis.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10773716/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139405435","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":"Enabling genome editing in tropical maize lines through an improved, morphogenic regulator-assisted transformation protocol","authors":"José Hernandes-Lopes, Maísa Siqueira Pinto, Letícia Rios Vieira, Patrícia Brant Monteiro, Sophia V. Gerasimova, Juliana Vieira Almeida Nonato, Maria Helena Faustinoni Bruno, Alexander Vikhorev, Fernanda Rausch-Fernandes, I. Gerhardt, L. Pauwels, Paulo Arruda, R. A. Dante, J. Yassitepe","doi":"10.3389/fgeed.2023.1241035","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1241035","url":null,"abstract":"The recalcitrance exhibited by many maize (Zea mays) genotypes to traditional genetic transformation protocols poses a significant challenge to the large-scale application of genome editing (GE) in this major crop species. Although a few maize genotypes are widely used for genetic transformation, they prove unsuitable for agronomic tests in field trials or commercial applications. This challenge is exacerbated by the predominance of transformable maize lines adapted to temperate geographies, despite a considerable proportion of maize production occurring in the tropics. Ectopic expression of morphogenic regulators (MRs) stands out as a promising approach to overcome low efficiency and genotype dependency, aiming to achieve ’universal’ transformation and GE capabilities in maize. Here, we report the successful GE of agronomically relevant tropical maize lines using a MR-based, Agrobacterium-mediated transformation protocol previously optimized for the B104 temperate inbred line. To this end, we used a CRISPR/Cas9-based construct aiming at the knockout of the VIRESCENT YELLOW-LIKE (VYL) gene, which results in an easily recognizable phenotype. Mutations at VYL were verified in protoplasts prepared from B104 and three tropical lines, regardless of the presence of a single nucleotide polymorphism (SNP) at the seed region of the VYL target site in two of the tropical lines. Three out of five tropical lines were amenable to transformation, with efficiencies reaching up to 6.63%. Remarkably, 97% of the recovered events presented indels at the target site, which were inherited by the next generation. We observed off-target activity of the CRISPR/Cas9-based construct towards the VYL paralog VYL-MODIFIER, which could be partly due to the expression of the WUSCHEL (WUS) MR. Our results demonstrate efficient GE of relevant tropical maize lines, expanding the current availability of GE-amenable genotypes of this major crop.","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-12-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138591509","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Progress in gene editing tools, implications and success in plants: a review","authors":"Suman Jyoti Bhuyan, Manoj Kumar, Pandurang Ramrao Devde, A. C. Rai, Amit Kumar Mishra, Prashant Kumar Singh, K. Siddique","doi":"10.3389/fgeed.2023.1272678","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1272678","url":null,"abstract":"Genetic modifications are made through diverse mutagenesis techniques for crop improvement programs. Among these mutagenesis tools, the traditional methods involve chemical and radiation-induced mutagenesis, resulting in off-target and unintended mutations in the genome. However, recent advances have introduced site-directed nucleases (SDNs) for gene editing, significantly reducing off-target changes in the genome compared to induced mutagenesis and naturally occurring mutations in breeding populations. SDNs have revolutionized genetic engineering, enabling precise gene editing in recent decades. One widely used method, homology-directed repair (HDR), has been effective for accurate base substitution and gene alterations in some plant species. However, its application has been limited due to the inefficiency of HDR in plant cells and the prevalence of the error-prone repair pathway known as non-homologous end joining (NHEJ). The discovery of CRISPR-Cas has been a game-changer in this field. This system induces mutations by creating double-strand breaks (DSBs) in the genome and repairing them through associated repair pathways like NHEJ. As a result, the CRISPR-Cas system has been extensively used to transform plants for gene function analysis and to enhance desirable traits. Researchers have made significant progress in genetic engineering in recent years, particularly in understanding the CRISPR-Cas mechanism. This has led to various CRISPR-Cas variants, including CRISPR-Cas13, CRISPR interference, CRISPR activation, base editors, primes editors, and CRASPASE, a new CRISPR-Cas system for genetic engineering that cleaves proteins. Moreover, gene editing technologies like the prime editor and base editor approaches offer excellent opportunities for plant genome engineering. These cutting-edge tools have opened up new avenues for rapidly manipulating plant genomes. This review article provides a comprehensive overview of the current state of plant genetic engineering, focusing on recently developed tools for gene alteration and their potential applications in plant research.","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-12-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138983763","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Germline ablation achieved via CRISPR/Cas9 targeting of <i>NANOS3</i> in bovine zygotes.","authors":"Maci L Mueller, Bret R McNabb, Joseph R Owen, Sadie L Hennig, Alba V Ledesma, Mitchell L Angove, Alan J Conley, Pablo J Ross, Alison L Van Eenennaam","doi":"10.3389/fgeed.2023.1321243","DOIUrl":"10.3389/fgeed.2023.1321243","url":null,"abstract":"<p><p><i>NANOS3</i> is expressed in migrating primordial germ cells (PGCs) to protect them from apoptosis, and it is known to be a critical factor for germline development of both sexes in several organisms. However, to date, live <i>NANOS3</i> knockout (KO) cattle have not been reported, and the specific role of <i>NANOS3</i> in male cattle, or bulls, remains unexplored. This study generated <i>NANOS3</i> KO cattle <i>via</i> cytoplasmic microinjection of the CRISPR/Cas9 system <i>in vitro</i> produced bovine zygotes and evaluated the effect of <i>NANOS3</i> elimination on bovine germline development, from fetal development through reproductive age. The co-injection of two selected guide RNA (gRNA)/Cas9 ribonucleoprotein complexes (i.e., dual gRNA approach) at 6 h post fertilization achieved a high <i>NANOS3</i> KO rate in developing embryos. Subsequent embryo transfers resulted in a 31% (<i>n</i> = 8/26) pregnancy rate. A 75% (<i>n</i> = 6/8) total KO rate (i.e., 100% of alleles present contained complete loss-of-function mutations) was achieved with the dual gRNA editing approach. In <i>NANOS3</i> KO fetal testes, PGCs were found to be completely eliminated by 41-day of fetal age. Importantly, despite the absence of germ cells, seminiferous tubule development was not impaired in <i>NANOS3</i> KO bovine testes during fetal, perinatal, and adult stages. Moreover, a live, <i>NANOS3</i> KO, germline-ablated bull was produced and at sexual maturity he exhibited normal libido, an anatomically normal reproductive tract, and intact somatic gonadal development and structure. Additionally, a live, <i>NANOS3</i> KO, germline-ablated heifer was produced. However, it was evident that the absence of germ cells in <i>NANOS3</i> KO cattle compromised the normalcy of ovarian development to a greater extent than it did testes development. The meat composition of <i>NANOS3</i> KO cattle was unremarkable. Overall, this study demonstrated that the absence of <i>NANOS3</i> in cattle leads to the specific deficiency of both male and female germ cells, suggesting the potential of <i>NANOS3</i> KO cattle to act as hosts for donor-derived exogenous germ cell production in both sexes. These findings contribute to the understanding of <i>NANOS3</i> function in cattle and have valuable implications for the development of novel breeding technologies using germline complementation in <i>NANOS3</i> KO germline-ablated hosts.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10711618/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138814211","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":"Reciprocal mutations of lung-tropic AAV capsids lead to improved transduction properties.","authors":"Ashley L Cooney, Christian M Brommel, Soumba Traore, Gregory A Newby, David R Liu, Paul B McCray, Patrick L Sinn","doi":"10.3389/fgeed.2023.1271813","DOIUrl":"10.3389/fgeed.2023.1271813","url":null,"abstract":"<p><p>Considerable effort has been devoted to developing adeno-associated virus (AAV)-based vectors for gene therapy in cystic fibrosis (CF). As a result of directed evolution and capsid shuffling technology, AAV capsids are available with widespread tropism for airway epithelial cells. For example, AAV2.5T and AAV6.2 are two evolved capsids with improved airway epithelial cell transduction properties over their parental serotypes. However, limited research has been focused on identifying their specific cellular tropism. Restoring cystic fibrosis transmembrane conductance regulator (<i>CFTR</i>) expression in surface columnar epithelial cells is necessary for the correction of the CF airway phenotype. Basal cells are a progenitor population of the conducting airways responsible for replenishing surface epithelial cells (including secretory cells and ionocytes), making correction of this cell population vital for a long-lived gene therapy strategy. In this study, we investigate the tropism of AAV capsids for three cell types in primary cultures of well-differentiated human airway epithelial (HAE) cells and primary human airway basal cells. We observed that AAV2.5T transduced surface epithelial cells better than AAV6.2, while AAV6.2 transduced airway basal cells better than AAV2.5T. We also investigated a recently developed capsid, AAV6.2FF, which has two surface tyrosines converted to phenylalanines. Next, we incorporated reciprocal mutations to create AAV capsids with further improved surface and basal cell transduction characteristics. Lastly, we successfully employed a split-intein approach using AAV to deliver an adenine base editor (ABE) to repair the <i>CFTR</i> ^R553X mutation. Our results suggest that rational incorporation of AAV capsid mutations improves AAV transduction of the airway surface and progenitor cells and may ultimately lead to improved pulmonary function in people with CF.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10702583/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138814216","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 editing for agricultural sustainability: developments in tools, potential applications, and regulatory policy.","authors":"Felicity J Keiper, Thorben Sprink, Ian Douglas Godwin","doi":"10.3389/fgeed.2023.1324921","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1324921","url":null,"abstract":"","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10644806/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138464768","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 gene editing to improve crop resistance to parasitic plants.","authors":"Min-Yao Jhu, Evan E Ellison, Neelima R Sinha","doi":"10.3389/fgeed.2023.1289416","DOIUrl":"10.3389/fgeed.2023.1289416","url":null,"abstract":"<p><p>Parasitic plants pose a significant threat to global agriculture, causing substantial crop losses and hampering food security. In recent years, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene-editing technology has emerged as a promising tool for developing resistance against various plant pathogens. Its application in combating parasitic plants, however, remains largely unexplored. This review aims to summarise current knowledge and research gaps in utilising CRISPR to develop resistance against parasitic plants. First, we outline recent improvements in CRISPR gene editing tools, and what has been used to combat various plant pathogens. To realise the immense potential of CRISPR, a greater understanding of the genetic basis underlying parasitic plant-host interactions is critical to identify suitable target genes for modification. Therefore, we discuss the intricate interactions between parasitic plants and their hosts, highlighting essential genes and molecular mechanisms involved in defence response and multilayer resistance. These include host resistance responses directly repressing parasitic plant germination or growth and indirectly influencing parasitic plant development via manipulating environmental factors. Finally, we evaluate CRISPR-mediated effectiveness and long-term implications for host resistance and crop improvement, including inducible resistance response and tissue-specific activity. In conclusion, this review highlights the challenges and opportunities CRISPR technology provides to combat parasitic plants and provides insights for future research directions to safeguard global agricultural productivity.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10642197/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"107593029","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":"Characterization of transcriptional enhancers in the chicken genome using CRISPR-mediated activation.","authors":"Jeong Hoon Han, Hong Jo Lee, Tae Hyun Kim","doi":"10.3389/fgeed.2023.1269115","DOIUrl":"10.3389/fgeed.2023.1269115","url":null,"abstract":"<p><p>DNA regulatory elements intricately control when, where, and how genes are activated. Therefore, understanding the function of these elements could unveil the complexity of the genetic regulation network. Genome-wide significant variants are predominantly found in non-coding regions of DNA, so comprehending the predicted functional regulatory elements is crucial for understanding the biological context of these genomic markers, which can be incorporated into breeding programs. The emergence of CRISPR technology has provided a powerful tool for studying non-coding regulatory elements in genomes. In this study, we leveraged epigenetic data from the Functional Annotation of Animal Genomes project to identify promoter and putative enhancer regions associated with three genes (<i>HBBA, IRF7</i>, and <i>PPARG</i>) in the chicken genome. To identify the enhancer regions, we designed guide RNAs targeting the promoter and candidate enhancer regions and utilized CRISPR activation (CRISPRa) with dCas9-p300 and dCas9-VPR as transcriptional activators in chicken DF-1 cells. By comparing the expression levels of target genes between the promoter activation and the co-activation of the promoter and putative enhancers, we were able to identify functional enhancers that exhibited augmented upregulation. In conclusion, our findings demonstrate the remarkable efficiency of CRISPRa in precisely manipulating the expression of endogenous genes by targeting regulatory elements in the chicken genome, highlighting its potential for functional validation of non-coding regions.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10634339/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89720934","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 mutagenesis of the <i>Arabidopsis</i> GROWTH-REGULATING FACTOR (GRF) gene family.","authors":"Juan Angulo,&nbsp;Christopher P Astin,&nbsp;Olivia Bauer,&nbsp;Kelan J Blash,&nbsp;Natalee M Bowen,&nbsp;Nneoma J Chukwudinma,&nbsp;Austin S DiNofrio,&nbsp;Donald O Faletti,&nbsp;Alexa M Ghulam,&nbsp;Chloe M Gusinde-Duffy,&nbsp;Kamaria J Horace,&nbsp;Andrew M Ingram,&nbsp;Kylie E Isaack,&nbsp;Geon Jeong,&nbsp;Randolph J Kiser,&nbsp;Jason S Kobylanski,&nbsp;Madeline R Long,&nbsp;Grace A Manning,&nbsp;Julie M Morales,&nbsp;Kevin H Nguyen,&nbsp;Robin T Pham,&nbsp;Monthip H Phillips,&nbsp;Tanner W Reel,&nbsp;Jenny E Seo,&nbsp;Hiep D Vo,&nbsp;Alexander M Wukoson,&nbsp;Kathryn A Yeary,&nbsp;Grace Y Zheng,&nbsp;Wolfgang Lukowitz","doi":"10.3389/fgeed.2023.1251557","DOIUrl":"https://doi.org/10.3389/fgeed.2023.1251557","url":null,"abstract":"<p><p>Genome editing in plants typically relies on T-DNA plasmids that are mobilized by <i>Agrobacterium</i>-mediated transformation to deliver the CRISPR/Cas machinery. Here, we introduce a series of CRISPR/Cas9 T-DNA vectors for minimal settings, such as teaching labs. Gene-specific targeting sequences can be inserted as annealed short oligonucleotides in a single straightforward cloning step. Fluorescent markers expressed in mature seeds enable reliable selection of transgenic or transgene-free individuals using a combination of inexpensive LED lamps and colored-glass alternative filters. Testing these tools on the <i>Arabidopsis</i> GROWTH-REGULATING FACTOR (GRF) genes, we were able to create a collection of predicted null mutations in all nine family members with little effort. We then explored the effects of simultaneously targeting two, four and eight GRF genes on the rate of induced mutations at each target locus. In our hands, multiplexing was associated with pronounced disparities: while mutation rates at some loci remained consistently high, mutation rates at other loci dropped dramatically with increasing number of single guide RNA species, thereby preventing a systematic mutagenesis of the family.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10613670/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"71429844","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}