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Current and Future Therapeutic Strategies for Lentiviral Eradication from Macrophage Reservoirs.
Macrophages, one of the most abundant populations of leukocytes in the body, function as the first line of defense against pathogen invaders. Human Immunodeficiency virus 1 (HIV-1) remains to date one of the most extensively studied viral infections. Naturally occurring lentiviruses in domestic and primate species serve as valuable models to investigate lentiviral pathogenesis and novel therapeutics. Better understanding of the role macrophages play in HIV pathogenesis will aid in the advancement towards a cure. Even with current efficacy of first- and second-line Antiretroviral Therapy (ART) guidelines and future efficacy of Long Acting Slow Effective Release-ART (LASER-ART); ART alone does not lead to a cure. The major challenge of HIV eradication is viral latency. Latency Reversal Agents (LRAs) show promise as a possible means to eradicate HIV-1 from the body. It has become evident that complete eradication will need to include combinations of various effective therapeutic strategies such as LASER-ART, LRAs, and gene editing. Review of the current literature indicates the most promising HIV eradication strategy appears to be LASER-ART in conjunction with viral and receptor gene modifications via the CRISPR/Cas9 system. Graphical abstract A multimodal approach to HIV treatment including gene editing, LASER-ART, and latency reversal agents may provide a means to achieve HIV eradication.
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Vulnerability and the Ethics of Human Germline Genome Editing.
The concept of vulnerability has played an important role in theoretical bioethics as well as in numerous authoritative guidelines on research ethics. The concept helps to identify situations in which research participants and other individuals may be at a heightened risk of experiencing harm. However, existing guidance documents on the ethics of human germline gene editing largely fail to make any reference to considerations of vulnerability. In this article, we discuss this oversight and we highlight the role that vulnerability can play in ethical debates about human heritable genome editing. Future guidance documents on germline gene editing should pay attention to considerations of vulnerability and reference these appropriately.
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CRISPR fixes disease gene in viable human embryos.
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Genome editing in Penicillium funiculosum using in vitro assembled CRISPR-Cas9 ribonucleoprotein complexes.
The plasmid-free CRISPR-Cas9-based genome editing in fungi is a precise and time-saving approach. Here, we present a detailed protocol for genetic manipulation in Penicillium funiculosum, which includes design and synthesis of sgRNA, high-quality protoplast preparation, and PEG-mediated protoplast transformation of linear donor DNA along with in vitro synthesized RNP complex composed of sgRNA and host-specific Cas9. This technique is beneficial for researchers interested in functional analysis of genes as it improves reproducibility and replicability of the experiment. For complete details on the use and execution of this protocol, please refer to Randhawa et al. (2021).
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CRISPR/Cas9-Mediated Genome Editing in Indica Rice (Oryza sativa L. subsp. indica var. CR-5272).
Tissue culture optimization protocols limit indica rice breeding. Such a challenge is vital because emergent techniques still rely on tissue culture methods and could allow the breeding of new varieties with higher production and toleration of adverse environmental effects caused by climate change. Genome editing technology, using CRISPR/Cas9, is a fast and precise method for accelerated plant breeding. It limited its use in indica subspecies because of the recalcitrant response to in vitro culture methods. This chapter describes a protocol for CRISPR/Cas9 editing in indica subspecies, specifically in the CR-5272 variety derived from parental lines IR-822, using Agrobacterium tumefaciens and biolistic transformation.
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Exosome-mediated horizontal gene transfer occurs in double-strand break repair during genome editing.
The CRISPR-Cas9 system has been successfully applied in many organisms as a powerful genome-editing tool. Undoubtedly, it will soon be applied to human genome editing, including gene therapy. We have previously reported that unintentional DNA sequences derived from retrotransposons, genomic DNA, mRNA and vectors are captured at double-strand breaks (DSBs) sites when DSBs are introduced by the CRISPR-Cas9 system. Therefore, it is possible that unintentional insertions associated with DSB repair represent a potential risk for human genome editing gene therapies. To address this possibility, comprehensive sequencing of DSB sites was performed. Here, we report that exosome-mediated horizontal gene transfer occurs in DSB repair during genome editing. Exosomes are present in all fluids from living animals, including seawater and breathing mammals, suggesting that exosome-mediated horizontal gene transfer is the driving force behind mammalian genome evolution. The findings of this study highlight an emerging new risk for this leading-edge technology.
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Genome editing.
Recent advances in genome editing technologies have redefined our ability to probe and precisely edit the human genome and epigenome in vitro and in vivo. More specifically, RNA-guided CRISPR/Cas systems have revolutionized the field due to their simplicity in design and adaptability across biological systems. This Collection highlights results in CRISPR/Cas technology that increase the efficiency of precision genome editing, and allow genetic manipulation in model systems traditionally intractable to site-directed gene modification.
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The DreAM-plus integrative RNA switch enhances transient AAV expression and reduces side effects of gene editing.
Uncontrolled long-term adeno-associated virus (AAV) expression prohibits therapeutic strategies that require more precise and dynamic regulation. For example, long-lasting expression of gene editors by AAV could augment off-target effects and immunogenicity. Drug-inducible RNA switches are desirable tools to achieve transient AAV expression. However, current RNA switches only target a single mechanism such as transcription or RNA splicing, exhibiting limited capacity in transgene regulation. Here, we report DreAM-plus, a multilayer RNA switch that integrates an aptamer-based poly(A) regulator (pA), a drug-elicitable alternative splicing module (DreAM), and an engineered P2A element with conditional upstream open reading frames (uORFs). The pA-DreAM concatenation enhanced gene inducibility by up to 5-fold more than pA or DreAM alone, with 1.4- to 6.3-fold further improvement by uORFs. DreAM-plus achieved transient expression of an array of gene editors (SpCas9, SaCas9, Un1Cas12f1, OsCas12f1, AcCas12n, and IsDra2 TnpB) with a temporal resolution of less than 24 h, which significantly mitigated off-target effects by 1.4- to 2.8-fold. With lipid-nanoparticle-delivered pre-existing immunity in mice, DreAM-plus attenuated AAV-delivered Cas-specific CD8+ T cell immune toxicity in the liver and heart. Therefore, the inducible RNA switches could be synergistically integrated to build sophisticated genetic cassettes for enhanced safety of AAV-mediated gene editing.
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3'-end ligation sequencing is a sensitive method to detect DNA nicks at potential sites of off-target activity induced by prime editors.
Gene editing makes precise changes in DNA to restore normal function or expression of genes; however, the advancement of gene editing to the clinic is limited by the potential genotoxicity of off-target editing. To comprehensively identify potential sites in the genome that may be recognized by gene editing agents, in vitro approaches, in which the editor is combined with human genomic DNA and sites where editing may occur are identified biochemically, are important tools. Existing biochemical approaches for off-target discovery recognize double-stranded breaks generated by nuclease-based gene editors such as SpCas9, but novel approaches are needed for new editing modalities, such as prime editing, that nick one strand of DNA. To fill this gap, we have developed 3'-end ligation sequencing (PEG-seq), which can identify prime editor-induced nicks throughout the genome on in vitro digested human genomic DNA to identify potential off-target sites. Here we show that PEG-seq is an important addition to the off-target detection toolkit, enabling off-target discovery for DNA nicking gene editors such as prime editors.
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First Successful Targeted Mutagenesis Using CRISPR/Cas9 in Stably Transformed Grain Amaranth Tissue.
Grain amaranth is a nutritionally rich, stress-tolerant C4 dicot with considerable potential for climate-resilient agriculture; however, efficient and reproducible protocols for stable transformation, regeneration, and CRISPR/Cas9-mediated editing have not yet been established. CRISPR/Cas-based genome editing is a cornerstone technology for accelerating the development of climate-resilient, high-yielding crops. Its effective application depends on robust, stable transformation procedures and CRISPR/Cas systems optimised for the target species. The absence of such tools remains a critical constraint for the genetic improvement of many promising yet underexplored crops. In this study, we edited key genes of the betalain biosynthesis pathway in grain amaranth (Amaranthus hypochondriacus L.) using the CasCADE modular cloning system, thereby demonstrating the feasibility of targeted mutagenesis in an orphan crop. We observed successful edits in up to 49% of transformed calli, resulting in deletions or insertions in the target genes. Our CRISPR/Cas9-mediated editing paves the way for targeted molecular research and breeding of grain amaranth.
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