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European Court of Justice delivers no justice to Europe on genome-edited crops.
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Gene editing for Spinocerebellar ataxia type 3 taking advantage of the human ATXN3L paralog as replacement gene.
Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disease caused by a CAG expansion of the ataxin-3 gene (ATXN3). SCA3 patients suffer from ataxia, spasticity and dystonia in mid-adulthood, with spinocerebellar dysfunction and degeneration. As a monogenic disease for which only symptomatic treatment is available, ATXN3 is an attractive target for gene editing. We used the KamiCas9, a self-inactivating gene editing system, to explore gene editing strategies suitable for all SCA3 patients. We first tested the deletion of exon 10 or the introduction of a premature stop codon into exon 9. High editing events were observed in vitro, but efficiency was very low in SCA3 transgenic mice. We then evaluated an ablate-and-replace strategy. The ablate experiments resulted in 55 ± 18% cerebellar editing of the ATXN3 gene. A human ATXN3L paralog, expressed in the brains of SCA3 patients, may act as a natural, CRISPR-resistant replacement gene. In a proof-of-principle study, ablate and ablate-and-replace strategies were evaluated in SCA3 transgenic mice. Two months after injection, similar editing efficiencies were obtained in the ablate and ablate-and-replace groups. Immunofluorescence and RT-qPCR analyses of cerebellar markers support the development of this strategy for SCA3 treatment.
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Inverse pharmacology: Approaches and tools for introducing druggability into engineered proteins.
A major feature of twenty-first century medical research is the development of therapeutic strategies that use 'biologics' (large molecules, usually engineered proteins) and living cells instead of, or as well as, the small molecules that were the basis of pharmacology in earlier eras. The high power of these techniques can bring correspondingly high risk, and therefore the need for the potential for external control. One way of exerting control on therapeutic proteins is to make them responsive to small molecules; in a clinical context, these small molecules themselves have to be safe. Conventional pharmacology has resulted in thousands of small molecules licensed for use in humans, and detailed structural data on their binding to their protein targets. In principle, these data can be used to facilitate the engineering of drug-responsive modules, taken from natural proteins, into synthetic proteins. This has been done for some years (for example, Cre-ERT2) but usually in a painstaking manner. Recently, we have developed the bioinformatic tool SynPharm to facilitate the design of drug-responsive proteins. In this review, we outline the history of the field, the design and use of the Synpharm tool, and describe our own experiences in engineering druggability into the Cpf1 effector of CRISPR gene editing.
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Target-Specific Single Guide RNA (sgRNA) Design and In Vitro Validation of Target-Specific sgRNAs for CRISPR/Cas9-Mediated Editing of a Plant Stress Memory-Associated Gene.
CRISPR/Cas9 genome editing has emerged as a transformative tool in plant biology, enabling precise manipulation of genes involved in stress responses. In the context of plant stress memory, where prior exposure to environmental stress enhances subsequent stress tolerance. CRISPR-based approaches offer a powerful means to dissect and engineer underlying regulatory genes. A critical factor determining the success of CRISPR/Cas9 editing is the careful design and validation of single guide RNAs (sgRNAs), which guide the Cas9 nuclease to specific genomic targets. This chapter provides a detailed, step-by-step protocol for the design, in vitro transcription, and in vitro cleavage assay to check efficiency of target-specific sgRNAs for plant genome editing applications. As a case study, we describe the design and validation of sgRNAs targeting the Arabidopsis thaliana DREB2A gene, a key transcription factor associated with drought stress memory. Emphasis is placed on strategies to maximize on-target efficiency, minimize off-target effects, and assess sgRNA functionality in vitro prior to in planta applications. This chapter serves as a practical guide for researchers aiming to functionally characterize stress memory-associated genes using CRISPR/Cas9 technology.
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Controversial CRISPR 'gene drives' tested in mammals for the first time.
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Genome editing reagent delivery in plants.
Genome editing holds the potential for rapid crop improvement to meet the challenge of feeding the planet in a changing climate. The delivery of gene editing reagents into the plant cells has been dominated by plasmid vectors delivered using agrobacterium or particle bombardment. This approach involves the production of genetically engineered plants, which need to undergo regulatory approvals. There are various reagent delivery approaches available that have enabled the delivery of DNA-free editing reagents. They invariably involve the use of ribonucleoproteins (RNPs), especially in the case of CRISPR/Cas9-mediated gene editing. The explant of choice for most of the non-DNA approaches utilizes protoplasts as the recipient explant. While the editing efficiency is high in protoplasts, the ability to regenerate individual plants from edited protoplasts remains a challenge. There are various innovative delivery approaches being utilized to perform in planta edits that can be incorporated in the germline cells or inherited via seed. With the modification and adoption of various novel approaches currently being used in animal systems, it seems likely that non-transgenic genome editing will become routine in higher plants.
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Protocol for DNA Methylation Editing of Imprinted Loci and Assessment of the Effects.
In this chapter, we present an experimental protocol to conduct DNA methylation editing experiments, that is, to induce loss or gain of DNA methylation, targeting Dlk1-Dio3 imprinted domain, a well-studied imprinted locus, in ES cells. In this protocol, plasmid vectors expressing the DNA methylation editing tools, combining the CRISPR/dCas9 system and the SunTag system coupled to a DNA methyltransferase or a TET enzyme, are introduced into cells for transient expression. By employing this strategy, researchers can effectively investigate a distinct DNA methylation signature that has an impact on the imprinting status, including gene expression and histone modifications, across the entire domain. We also describe strategies for allele-specific quantitative analyses of DNA methylation, gene expression, and histone modifications and binding protein levels for assessing the imprinting state of the locus.
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[Back from Hong Kong or ethics at the time of a genetic increase of the human person].
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Unlocking the Synthetic Potential of Yarrowia lipolytica: Innovating Gene Expression Tools.
Yarrowia lipolytica, with its robust lipid metabolism capabilities, efficient secretion system and generally recognised as safe (GRAS) status, has become a highly promising microbial chassis in synthetic biology. However, compared with model microorganisms such as Saccharomyces cerevisiae, the underdevelopment of gene expression tools in Y. lipolytica has become a critical bottleneck, limiting its industrial application. Currently, its core tools face two critical challenges: promoters with limited dynamic regulatory capacity, leading to metabolic flux imbalance; and gene editing systems plagued by low efficiency and poor multiplex compatibility. This opinion article focuses on these two pivotal directions to dissect their technical bottlenecks and propose innovative solutions: constructing dynamic transcriptional regulatory modules through machine learning guided design and synthetic biology approaches and developing orthogonal CRISPR systems and multiplex editing platforms.
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After the Storm - A Responsible Path for Genome Editing.
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