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Editing the kinesin-12 gene affects responses to Bt toxin Cry1Ac in Helicoverpa zea.
Crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt) are used globally to manage key insect pests. However, the evolution of resistance to Bt proteins in at least 11 pest species has reduced the effectiveness of Bt crops. Resistance to crystalline (Cry) Bt proteins including Cry1Ac produced by Bt cotton is a major problem in Helicoverpa zea (also known as bollworm and corn earworm), one of the most economically damaging pests in the United States. A previous genome-wide association study identified a nonsense point mutation in a kinesin-12 gene that was associated with resistance to Cry1Ac in a lab-selected strain of H. zea. Here, we used CRISPR/Cas9 gene editing to knock out the kinesin-12 gene in a Cry1Ac-susceptible laboratory strain, which caused a 4.0-fold increase in resistance to Cry1Ac. Conversely, gene editing that repaired the natural kinesin-12 nonsense mutation in a lab-selected resistant strain increased susceptibility to Cry1Ac by 3.8-fold. These complementary results provide compelling evidence that kinesin-12 plays a role in the mode of action of Cry1Ac against H. zea.
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Generation of conditional Acvrl1 knockout mice by CRISPR/Cas9-mediated gene targeting.
OBJECTIVES: This study aimed to generate mutant mice containing the Acvrl1 gene flanked with LoxP sequences to allow conditional deletion of Acvrl1 by the LoxP/Cre system. Such mice may facilitate the development of brain arteriovenous malformation (BAVM) models. METHODS: The CRISPR/Cas9 technique was used to edit Acvrl1. Two single guide RNAs (sgRNAs) with recognition sites on intron 3 and 8 and a donor vector that was homologous with the targeted gene and contained two LoxP sequences were designed and constructed. The in vitro-synthesized sgRNA, Cas9 mRNA and donor vectors were injected into mouse zygotes, which were then transferred into pseudopregnant mice. Neonatal mutant mice were identified by genotyping and sequencing. RESULTS: Two mice with a floxed Acvrl1 allele were generated at a success rate of 8.7%. The target mice, which were healthy and fertile, were obtained through interbreeding. CONCLUSION: CRISPR/Cas9 is a reliable gene-editing tool, and is able to efficiently modify Acvrl1 and create the target mice.
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Restoring the Balance: CRISPRa-Driven β-Tubulin Compensation as a Strategy for Tubulinopathy Treatment.
Microtubules are essential cytoskeletal components comprising alpha- and beta-tubulin proteins that facilitate organelle positioning, cell migration, division, and intracellular trafficking. Mutations in tubulin genes can lead to tubulinopathies, a class of rare genetic neurodevelopmental disorders characterized by a range of brain malformations and other clinical features. Recent studies have shown that pathogenic variants in beta-tubulin genes such as TUBBG308S have been found to underlie the development of ciliopathies, disorders impacting cilia, important organelles for development and cell motility. Thus, mutations in distinct tubulin genes, which present a significant hurdle for the development of therapeutic gene editing strategies and targeted therapeutics. Here, we describe the development of a mutation-independent treatment strategy based on the upregulation of non-mutated beta-tubulin isotype protein using CRISPR-Cas9 activation. By increasing the expression of various beta-tubulin proteins, we demonstrate a restoration of the microtubule network and primary cilia formation.
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Genetics experience impacts attitudes towards germline gene editing: a survey of over 1500 members of the public.
CRISPR-Cas9 has revolutionised genome engineering and has the potential to radically change our approach to genetic disease. However, the potential for genetic modification of embryos has raised significant and complex ethical and social concerns. The scientific community have called for ongoing stakeholder consultation about Germline Gene Editing (GGE), in particular lay publics, to help guide policy, education, research and regulatory priorities. In response, we conducted a survey to gauge public support for GGE and describe the demographic, experiential and contextual factors that influence individual attitudes. Respondent support was measured across nine hypothetical medical and enhancement GGE applications. We received responses from 1537 participants across 67 countries. Respondents were generally supportive of GGE, particularly for medical applications. While the most opposition observed was among religious respondents, those with work experience in genetics or genomics also reported greater resistance to GGE. Personal or family-related experience with genetics or genomics, identifying as female and tertiary education were also associated with less support for GGE. Further research needs to explore a diverse range of community and group attitudes towards GGE; the reasons underlying demographic and experiential differences; and to determine where the public and genetics professionals draw the line on ethical implementation respectively.
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Identification of Ziziphus jujuba cv. Dongzao DNA Demethylase ZjROS1 Gene Family and Construction of CRISPR/Cas9-Mediated Gene-Editing Vector.
DNA methylation is one of the earliest and most extensively studied epigenetic regulatory mechanisms. The ROS1 (Repressor of Silencing 1) gene was first discovered in Arabidopsis thaliana, and it is a DNA demethylase that can remove 5-methylcytosine from DNA, thereby affecting DNA methylation levels and gene expression. Objectives: The objective of this study was to investigate the role of ROS1 in the development and maturation of Ziziphus jujuba cv. "Dongzao" fruit. Methods: We cloned the ROS1 gene and conducted bioinformatics and expression characteristics analyses on it. Results: Three ROS1 genes, named ZjROS1-1~3, was identified, and each member protein was localized in the nucleus, cytoskeleton, chloroplast, and vacuole. The promoter contained cis-elements such as light response, plant hormone signal transduction, and stress response cis-elements, and it interacted with many proteins such as CMT, MET, and ZDP. The results of the real-time fluorescence quantitative PCR show that ZjROS1 has specific expression patterns in different tissues of Z. jujuba cv. Dongzao, and the expression of ZjROS1-2 in flowers and fruits is high. At the same time, CRISPR/Cas9 technology was used to construct a gene-editing vector for ZjROS1, which provided a basis for the subsequent genetic transformation. Conclusions: In this study, the biological function of ZjROS1 was clarified and a gene-editing vector was constructed, which provided a theoretical basis for the regulation mechanism of demethylase ZjROS1 in the fruit ripening and development of Z. jujuba cv. Dongzao.
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Identification of a Novel Cleavage Site and Confirmation of the Effectiveness of NgAgo Gene Editing on RNA Targets.
Clusters of regularly interspaced short palindromic repeats (CRISPR)/Cas systems have a powerful ability to edit DNA and RNA targets. However, the need for a specific recognition site, protospacer adjacent motif (PAM), of the CRISPR/Cas system limits its application in gene editing. Some Argonaute (Ago) proteins have endonuclease functions under the guidance of 5' phosphorylated or hydroxylated guide DNA (gDNA). The NgAgo protein might perform RNA gene editing at 37 °C, suggesting its application in mammalian cells; however, its mechanisms are unclear. In the present study, the target of NgAgo in RNA was confirmed in vitro and in vivo. Then, an in vitro RNA cleavage system was designed and the cleavage site was verified by sequencing. Furthermore, NgAgo and gDNA were transfected into cells to cleave an intracellular target sequence. We demonstrated targeted degradation of GFP, HCV, and AKR1B10 RNAs in a gDNA-dependent manner by NgAgo both in vitro and in vivo, but no effect on DNA was observed. Sequencing demonstrated that the cleavage sites are located at the 3' of the target RNA which is recognized by 5' sequence of the gDNA. These results confirmed that NgAgo-gDNA cleaves RNA not DNA. We observed that the cleavage site is located at the 3' of the target RNA, which is a new finding that has not been reported in the past.
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Allele-specific genome editing using CRISPR-Cas9 is associated with loss of heterozygosity in diploid yeast.
Targeted DNA double-strand breaks (DSBs) with CRISPR-Cas9 have revolutionized genetic modification by enabling efficient genome editing in a broad range of eukaryotic systems. Accurate gene editing is possible with near-perfect efficiency in haploid or (predominantly) homozygous genomes. However, genomes exhibiting polyploidy and/or high degrees of heterozygosity are less amenable to genetic modification. Here, we report an up to 99-fold lower gene editing efficiency when editing individual heterozygous loci in the yeast genome. Moreover, Cas9-mediated introduction of a DSB resulted in large scale loss of heterozygosity affecting DNA regions up to 360 kb and up to 1700 heterozygous nucleotides, due to replacement of sequences on the targeted chromosome by corresponding sequences from its non-targeted homolog. The observed patterns of loss of heterozygosity were consistent with homology directed repair. The extent and frequency of loss of heterozygosity represent a novel mutagenic side-effect of Cas9-mediated genome editing, which would have to be taken into account in eukaryotic gene editing. In addition to contributing to the limited genetic amenability of heterozygous yeasts, Cas9-mediated loss of heterozygosity could be particularly deleterious for human gene therapy, as loss of heterozygous functional copies of anti-proliferative and pro-apoptotic genes is a known path to cancer.
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In vivo genome editing for inherited retinal disease: Opportunities and challenges.
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Prospects and challenges of in vivo hematopoietic stem cell genome editing for hemoglobinopathies.
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eVLP compound delivery breaks the prime editing efficiency ceiling.
Prime editing could resolve gene variant function at scale, but the editing machinery needs to be delivered efficiently and reproducibly. Langley, Baudrier, et al.1 show that timing of editor delivery is rate limiting and introduce PRIME-VLP, a repeated dosing strategy with engineered virus-like particles that improves editing efficiency and screening performance.
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