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Creation of an albino squid line by CRISPR-Cas9 and its application for in vivo functional imaging of neural activity.
Cephalopods are remarkable among invertebrates for their cognitive abilities, adaptive camouflage, novel structures, and propensity for recoding proteins through RNA editing. Due to the lack of genetically tractable cephalopod models, however, the mechanisms underlying these innovations are poorly understood. Genome editing tools such as CRISPR-Cas9 allow targeted mutations in diverse species to better link genes and function. One emerging cephalopod model, Euprymna berryi, produces large numbers of embryos that can be easily cultured throughout their life cycle and has a sequenced genome. As proof of principle, we used CRISPR-Cas9 in E. berryi to target the gene for tryptophan 2,3 dioxygenase (TDO), an enzyme required for the formation of ommochromes, the pigments present in the eyes and chromatophores of cephalopods. CRISPR-Cas9 ribonucleoproteins targeting tdo were injected into early embryos and then cultured to adulthood. Unexpectedly, the injected specimens were pigmented, despite verification of indels at the targeted sites by sequencing in injected animals (G0s). A homozygote knockout line for TDO, bred through multiple generations, was also pigmented. Surprisingly, a gene encoding indoleamine 2,3, dioxygenase (IDO), an enzyme that catalyzes the same reaction as TDO in vertebrates, was also present in E. berryi. Double knockouts of both tdo and ido with CRISPR-Cas9 produced an albino phenotype. We demonstrate the utility of these albinos for in vivo imaging of Ca2+ signaling in the brain using two-photon microscopy. These data show the feasibility of making gene knockout cephalopod lines that can be used for live imaging of neural activity in these behaviorally sophisticated organisms.
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Governing Heritable Human Genome Editing: A Textual History and a Proposal for the Future.
Heritable human genome editing (HHGE) has become a topic of intense public interest, especially since 2015. In the early 1980s, a related topic-human genetic engineering-was the subject of sustained public discussion. There was particular concern about germline genetic intervention. During the 1980s debate, an advisory committee to the Director of the National Institutes of Health (NIH)-the Recombinant DNA Advisory Committee (RAC)-agreed to provide initial public review of proposals for deliberate introduction of DNA into human beings. In 1984 and 1985, the RAC developed guidelines for research involving DNA transfer into patients. The committee also commented on the possibility of deliberately altering the human germline. We track the textual changes over time in the RAC's response to the possibility of germline genetic intervention in humans. In 2019, the NIH RAC was abolished. New techniques for genome editing, including CRISPR-based techniques, make both somatic and germline alterations much more feasible. These novel capabilities have again raised questions about oversight. We propose the creation of a new structure for the public oversight of proposals to perform HHGE. In parallel with a technical review by a regulatory agency, such proposals should also be publicly evaluated by a presidentially appointed Bioethics Advisory Commission.
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Zygote origin shapes sensitivity to electrical voltage field and editing susceptibility after CRISPR/Cas9-RNP electroporation in bovine embryos.
Despite major progress in generation of edited embryos and offspring via CRISPR/Cas9-RNP electroporation, editing outcomes remain highly variable. This variability may be due to intrinsic differences between in vivo-derived (IVV) and in vitro-derived (IVT) zygotes, which may play a more decisive role than previously anticipated. Therefore, this study aimed to determine how zygote origin shapes the sensitivity to electrical voltage field and editing susceptibility after CRISPR/Cas9-RNP electroporation in the bovine model. The present study systematically compared subsequent developmental competence and editing events in IVV- and IVT-derived bovine zygotes across defined electroporation voltages and pulse durations. Notably, CRISPR/Cas9-RNP electroporation of IVV-derived bovine zygotes of comparable age and collected by tubal embryo flushing (10-18 h post ovulation, hpo) resulted in markedly lower survival rates at 25 and 30 V compared to 20 V. In contrast, higher (p < 0.05) editing frequencies were observed at 25 V and 30 V. Clear differences, however, were observed between the two zygote groups. Following CRISPR/Cas9 RNP electroporation, IVV-derived zygotes yielded a higher proportion of unedited blastocysts (p < 0.05), absence of full edited embryos, a significantly increased frequency of monoallelic edits and reduced rates of mosaicism compared to IVT-derived zygotes. Collectively, the results of the present study clearly underscore a significant lower editing efficiency to pulse sensitivity ratio in IVV-derived zygotes compared to their IVT counterparts. To our knowledge, this is the first study to use bovine IVV-derived zygotes for genome editing using RNP-electroporation to elucidate how zygote origin shapes sensitivity to electrical voltage field and editing efficiency.
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Hopes and Difficulties for Blastocyst Complementation.
The clinical need for organ replacement therapies has inspired the idea of growing human organs in animal hosts. The injection of human pluripotent stem cells into animal blastocysts provides a possible strategy to accomplish this goal. Subject of Review: A recent study [Wu et al. Cell 2017;168:473-486.e415] tests the feasibility of this approach by creating chimeric embryos between humans and large domestic animals, including pigs and cattle. The study further examines the potential of combining CRISPR-Cas9 gene editing with blastocyst complementation to grow fully foreign organs in chimeric hosts. Second Opinion: Here, we consider what this report and related studies reveal about the likelihood of human-animal chimeras reaching the clinic and translating into therapies. A careful look suggests hope for eventual success in this area but also underscores important challenges that will require dedicated effort to resolve.
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Autologous gene therapy for hemoglobinopathies: From bench to patient's bedside.
In recent years, a growing number of clinical trials have been initiated to evaluate gene therapy approaches for the treatment of patients with transfusion-dependent β-thalassemia and sickle cell disease (SCD). Therapeutic modalities being assessed in these trials utilize different molecular techniques, including lentiviral vectors to add functional copies of the gene encoding the hemoglobin β subunit in defective cells and CRISPR-Cas9, transcription activator-like effector protein nuclease, and zinc finger nuclease gene editing strategies to either directly address the underlying genetic cause of disease or induce fetal hemoglobin production by gene disruption. Here, we review the mechanisms of action of these various gene addition and gene editing approaches and describe the status of clinical trials designed to evaluate the potentially for these approaches to provide one-time functional cures to patients with transfusion-dependent β-thalassemia and SCD.
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Toward a logic-based framework for plant epigenetic control.
Genome editing enables precise sequence alteration, but remains limited by binary logic and irreversible outcomes. By contrast, epigenome editing offers reversible and multilayered regulation without altering the DNA sequence. Yet current implementations remain inert - unable to sense, compute, or adapt. Here, we survey emerging plant epigenome editing modalities and explore their integration with logic-based synthetic gene circuits. We propose design strategies, such as multiplexer-driven flowering switches in Arabidopsis (Arabidopsis thaliana) and Boolean logic-gated fruit ripening in Solanum lycopersicum. Underpinned by plant-tailored roadmaps and pitfall mitigation strategies synthesized here, these architectures could transform static editing into programmable, context-aware regulation. This convergence gestures toward a future of composite epigenome engineering, where epigenetic plasticity and synthetic logic integrate to support scalable, predictive control of traits.
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CRISPR-Cas9 genome editing approaches in filamentous fungi and oomycetes.
Due to their biotechnological relevance as well as their importance as disease agents, filamentous fungi and oomycetes have been prime candidates for genetic selection and in vitro manipulation for decades. With the advent of new genome editing technologies such manipulations have reached a new level of speed and sophistication. The CRISPR-Cas9 genome editing technology in particular has revolutionized the ways how desired mutations can be introduced. To date, the CRISPR-Cas9 genome editing system has been established in more than 40 different species of filamentous fungi and oomycetes. In this review we describe the various approaches taken to assure expression of the components necessary for editing and describe the varying strategies used to achieve gene disruptions, gene replacements and precise editing. We discuss potential problems faced when establishing the system, propose ways to circumvent them and suggest future approaches not yet realized in filamentous fungi or oomycetes.
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Preclinical safety and biodistribution of CRISPR targeting SIV in non-human primates.
In this study, we demonstrate the safety and utility of CRISPR-Cas9 gene editing technology for in vivo editing of proviral DNA in ART-treated, virally controlled simian immunodeficiency virus (SIV) infected rhesus macaques, an established model for HIV infection. EBT-001 is an AAV9-based vector delivering SaCas9 and dual guide RNAs designed to target multiple regions of the SIV genome: the viral LTRs, and the Gag gene. The results presented here demonstrate that a single IV inoculation of EBT-001 at each of 3 dose levels (1.4 × 1012, 1.4 × 1013 and 1.4 × 1014 genome copies/kg) resulted in broad and functional biodistribution of AAV9-EBT-001 to known tissue reservoirs of SIV. No off-target effects or abnormal pathology were observed, and animals returned to their normal body weight after receiving EBT-001. Importantly, the macaques that received the 2 highest doses of EBT-001 showed improved absolute lymphocyte counts as compared to antiretroviral-treated controls. Taken together, these results demonstrate safety, biodistribution, and in vivo proviral DNA editing following IV administration of EBT-001, supporting the further development of CRISPR-based gene editing as a potential therapeutic approach for HIV in humans.
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Precise genome modification in tomato using an improved prime editing system.
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T7 Polymerase Expression of Guide RNAs in vivo Allows Exportable CRISPR-Cas9 Editing in Multiple Yeast Hosts.
Efficient guide RNA expression often limits CRISPR-Cas9 implementation in new hosts. To address this limitation in fungal systems, we demonstrate the utility of a T7 polymerase system to effectively express sgRNAs. Initially, we developed a methodology in Saccharomyces cerevisiae using a modified version of the T7 P266L mutant polymerase with an SV40 nuclear localization signal to allow guide RNA expression immediately downstream of a T7 promoter. To improve targeting efficiency, guide RNA design was found to be tolerant to three mismatches or up to three additional bases appended to the 5' end. The addition of three guanines to a T7-based guide RNA improved guide RNA expression 80-fold and achieved transcriptional output similar to the strong Pol III snr52 promoter. Resulting gene editing and dCas9-guided gene regulation with a T7-based guide RNA was on par with the commonly used snr52 system in S. cerevisiae. Finally, 96% and 60% genome editing efficiencies were achieved in Kluyveromyces lactis and Yarrowia lipolytica respectively with minimal optimization of this system. Thus, T7-based expression of sgRNAs offers an orthogonal method for implementing CRISPR systems in fungal systems.
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