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A pipeline for precise and efficient genome editing by sgRNA-Cas9 RNPs in Drosophila.
Genome editing via homology-directed repair (HDR) has made possible precise and deliberate modifications to gene sequences. CRISPR/Cas9-mediated HDR is the simplest means to carry this out. However, technical challenges remain to improve efficiency and broaden applicability to any genetic background of Drosophila melanogaster as well as to other Drosophila species. To address these issues, we developed a two-stage marker-assisted strategy in which embryos are injected with RNPs and pre-screened using T7EI. Using sgRNA in complex with recombinant Cas9 protein, we assayed each sgRNA for genome-cutting efficiency. We then conducted HDR using sgRNAs that efficiently cut target genes and the application of a transformation marker that generates RNAi against eyes absent. This allows for screening based on eye morphology rather than colour. These new tools can be used to make a single change or a series of allelic substitutions in a region of interest, or to create additional genetic tools such as balancer chromosomes.
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CRISPR-based gene editing for antimicrobial resistance control in human medicine.
Antimicrobial resistance (AMR) has already become one of the most urgent threats to the public health of this century. In 2019 alone, it directly causes about 1.27 million deaths and it was estimated that 1.91 million people will die yearly by 2050 should present trends persist. The traditional antibiotic development pipelines have been shown to be structurally insufficient to meet the rate at which bacterial populations have developed, diversified and spread resistance determinants, typically by horizontal gene transfer. In this context, CRISPR-Cas gene editing has become a focused antimicrobial approach that can selectively target resistance genes, virulence factors, and mobile genetic elements without the broad-spectrum collateral damage associated with conventional antibiotics. The review assesses CRISPR-Cas systems, namely Cas9, Cas12a, Cas3, and Cas13 in the context of two complementary mechanistic strategies namely selective killing of pathogens and antibiotic resensitization by the targeted disruption of gene resistance. We compare the impact of key delivery systems, such as bacteriophage vectors, lipid nanoparticles, and conjugative plasmids, evaluating them based on their therapeutic activity, host selectivity, and possible translation. The present state of clinical translations is discussed, including the two most advanced clinical-stage candidates SNIPR001 (Phase I/II, NCT05277350) and LBP-EC01 (Phase 2/3, NCT05488444). We also address the open issues that include off-target editing, host immune reactions, bacterial counter-resistance, regulatory ambiguity, and scalability of manufacturing. Lastly, we provide priority research directions, such as the combination antimicrobial strategies, AI-assisted CRISPR design, and next-generation delivery engineering, none of which will be resolved before routine clinical application of CRISPR-based antimicrobials is achieved.
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Neofunctionalization of a second insulin receptor gene in the wing-dimorphic planthopper, Nilaparvata lugens.
A single insulin receptor (InR) gene has been identified and extensively studied in model species ranging from nematodes to mice. However, most insects possess additional copies of InR, yet the functional significance, if any, of alternate InRs is unknown. Here, we used the wing-dimorphic brown planthopper (BPH) as a model system to query the role of a second InR copy in insects. NlInR2 resembled the BPH InR homologue (NlInR1) in terms of nymph development and reproduction, but revealed distinct regulatory roles in fuel metabolism, lifespan, and starvation tolerance. Unlike a lethal phenotype derived from NlInR1 null, homozygous NlInR2 null mutants were viable and accelerated DNA replication and cell proliferation in wing cells, thus redirecting short-winged-destined BPHs to develop into long-winged morphs. Additionally, the proper expression of NlInR2 was needed to maintain symmetric vein patterning in wings. Our findings provide the first direct evidence for the regulatory complexity of the two InR paralogues in insects, implying the functionally independent evolution of multiple InRs in invertebrates.
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ReMOT Control Delivery of CRISPR-Cas9 Ribonucleoprotein Complex to Induce Germline Mutagenesis in the Disease Vector Mosquitoes Culex pipiens pallens (Diptera: Culicidae).
The wide distribution of Culex (Cx.) pipiens complex mosquitoes makes it difficult to prevent the transmission of mosquito-borne diseases in humans. Gene editing using CRISPR/Cas9 is an effective technique with the potential to solve the growing problem of mosquito-borne diseases. This study uses the ReMOT Control technique in Culex pipiens pallens (L.) to produce genetically modified mosquitoes. A microinjection system was established by injecting 60 adult female mosquitoes-14 µl injection mixture was required, and no precipitation occurred with ≤1 µl of endosomal release reagents (chloroquine or saponin). The efficiency of delivery of the P2C-enhanced green fluorescent protein-Cas9 (P2C-EGFP-Cas9) ribonucleoprotein complex into the ovary was 100% when injected at 24 h post-bloodmeal (the peak of vitellogenesis). Using this method for KMO knockout, we found that gene editing in the ovary could also occur when P2C-Cas9 RNP complex was injected into the hemolymph of adult Cx. pipiens pallens by ReMOT Control. In the chloroquine group, of the 2,251 G0 progeny screened, 9 individuals showed with white and mosaic eye phenotypes. In the saponin group, of the 2,462 G0 progeny screened, 8 mutant individuals were observed. Sequencing results showed 13 bp deletions, further confirming the fact that gene editing occurred. In conclusion, the successful application of ReMOT Control in Cx. pipiens pallens not only provides the basic parameters (injection parameters and injection time) for this method but also facilitates the study of mosquito biology and control.
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"CRISPR for Disabilities: How to Self-Regulate" or Something?
The development of the CRISPR gene editing technique has been hyped as a technique that could fundamentally change scientific research and its clinical application. Unrecognized is the fact that it joins other technologies that have tried and failed under the same discourse of scientific hype. These technologies, like gene therapy and stem cell research, have moved quickly passed basic research into clinical application with dire consequences. Before hastily moving to clinical applications, it is necessary to consider basic research and determine how CRISPR/Cas systems should be applied. In the case of single gene diseases, that application is expected to have positive impacts, but as we shift to more complex diseases, the impact could be unintentionally negative. In the context of common disabilities, the level of genetic complexity may render this technology useless but potentially toxic, aggravating a social discourse that devalues those with disabilities. This paper intends to define the issues related to disability that are associated with using the CRIPSR/Cas system in basic research. It also aims to provide a decision tree to help determine whether the technology should be utilized or if alternative approaches beyond scientific research could lead to a better use of limited funding resources.
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CRISPR/Cas9 system: a powerful technology for in vivo and ex vivo gene therapy.
CRISPR/Cas9 is a versatile genome-editing tool which is widely used for modifying the genome of both prokaryotic and eukaryotic organisms for basic research and applications. An increasing number of reports have demonstrated that CRISPR/Cas9-mediated genome editing is a powerful technology for gene therapy. Here, we review the recent advances in CRISPR/Cas9-mediated gene therapy in animal models via different strategies and discuss the challenges as well as future prospects.
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Emerging DNA & RNA editing strategies for the treatment of epidermolysis bullosa.
Background: Epidermolysis bullosa (EB) is a clinically-heterogeneous genodermatosis with severe manifestations in the skin and other organs. The significant burden this condition places on patients justifies the development of gene therapeutic strategies targeting the genetic cause of the disease. Methods: Emerging RNA and DNA editing tools have shown remarkable advances in efficiency and safety. Applicable both in ex vivo- and in vivo settings, these gene therapeutics based on gene replacement or editing are either at the pre-clinical or clinical stage. Results: The recent landmark FDA approvals for gene editing based on CRISPR/Cas9, along with the first FDA-approved redosable in vivo gene replacement therapy for EB, will invigorate ongoing research efforts, increasing the likelihood of achieving local cure via CRISPR-based technologies in the near future. Conclusions: This review discusses the status quo of current gene therapeutics that act at the level of RNA or DNA, all with the common aim of improving the quality of life for EB patients.
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Synergizing CRISPR-Cas9 with Advanced Artificial Intelligence and Machine Learning for Precision Drug Delivery: Technological Nexus and Regulatory Insights.
The evolution of genetic exploration tools, from laborious methods like radiationinduced mutations to the transformative CRISPR-Cas9 system, has fundamentally reshaped genetic research and gene editing capabilities. This journey, initiated by foundational techniques such as ZFNs and TALENs and culminating in the groundbreaking work of Doudna and Charpentier in 2012, has ushered in an era of precise DNA alteration and profound insights into gene functions. The CRISPR/Cas9 system uses the Cas9 enzyme and guides RNA (gRNA) to precisely target and cleave DNA, with subsequent repair via error-prone NHEJ or precise HDR, enabling versatile gene editing. Complementary computational tools like E-CRISP and Azimuth 2.0, alongside advanced deep learning models like DeepCRISPR, have significantly contributed to refining CRISPR experiments, optimizing gRNA efficiency, and predicting outcomes with greater precision. In clinical applications, CRISPR-Cas9 shows great promise for treating complex genetic disorders like sickle cell disease and β-thalassemia, but faces challenges such as off-target effects, immune responses to viral vectors, and ethical issues in germline editing. Overcoming these challenges requires meticulous experimentation and robust regulatory frameworks to ensure responsible and beneficial utilization of the CRISPR-Cas9 technology across diverse fields, including cancer treatment, genetic disease therapies, agriculture, and synthetic biology, while continually addressing ethical, safety, and legal considerations for its advancement and widespread adoption.
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CRISPR/Cas9 in soybean research: a measured leap toward genetic refinement.
Soybean [Glycine max (Linn.) Merr] is a globally important source of plant-based protein and edible oil, widely cultivated in India as a major oilseed crop. However, conventional breeding approaches for soybean are laborious, constrained by the genomic complexity of soybean, and face persistent challenges in genetic transformation. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-based genome editing has emerged as a precise and efficient tool for rapid trait improvement in soybean. While many existing reviews of soybean have been published, this literature review addresses some fundamental challenges with respect to genome editing. This study summarizes recent advancements in soybean genome editing, offering insights into the practical informatics of soybean-specific genomic databases that are required for successful construct design. It also discusses emerging technologies such as base editing, prime editing, and the increasingly important role of artificial intelligence in accelerating target identification and construct design. It highlights applications in nutritional enhancement, abiotic and biotic stress tolerance, and improvement of key agronomic traits. Beyond the conventional topics, it addresses the current regulatory landscape that researchers must navigate for releasing genome-edited varieties and provides a comprehensive perspective on practical solutions to overcome major constraints in soybean genetic engineering.
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Application of genome-editing systems to enhance available pig resources for agriculture and biomedicine.
Traditionally, genetic engineering in the pig was a challenging task. Genetic engineering of somatic cells followed by somatic cell nuclear transfer (SCNT) could produce genetically engineered (GE) pigs carrying site-specific modifications. However, due to difficulties in engineering the genome of somatic cells and developmental defects associated with SCNT, a limited number of GE pig models were reported. Recent developments in genome-editing tools, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 system, have markedly changed the effort and time required to produce GE pig models. The frequency of genetic engineering in somatic cells is now practical. In addition, SCNT is no longer essential in producing GE pigs carrying site-specific modifications, because direct injection of genome-editing systems into developing embryos introduces targeted modifications. To date, the CRISPR/Cas9 system is the most convenient, cost-effective, timely and commonly used genome-editing technology. Several applicable biomedical and agricultural pig models have been generated using the CRISPR/Cas9 system. Although the efficiency of genetic engineering has been markedly enhanced with the use of genome-editing systems, improvements are still needed to optimally use the emerging technology. Current and future advances in genome-editing strategies will have a monumental effect on pig models used in agriculture and biomedicine.
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