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In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription.
Molecular barcoding technologies that uniquely identify single cells are hampered by limitations in barcode measurement. Readout by sequencing does not preserve the spatial organization of cells in tissues, whereas imaging methods preserve spatial structure but are less sensitive to barcode sequence. Here we introduce a system for image-based readout of short (20-base-pair) DNA barcodes. In this system, called Zombie, phage RNA polymerases transcribe engineered barcodes in fixed cells. The resulting RNA is subsequently detected by fluorescent in situ hybridization. Using competing match and mismatch probes, Zombie can accurately discriminate single-nucleotide differences in the barcodes. This method allows in situ readout of dense combinatorial barcode libraries and single-base mutations produced by CRISPR base editors without requiring barcode expression in live cells. Zombie functions across diverse contexts, including cell culture, chick embryos and adult mouse brain tissue. The ability to sensitively read out compact and diverse DNA barcodes by imaging will facilitate a broad range of barcoding and genomic recording strategies.
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CRISPR/Cpf1 multiplex genome editing system increases silkworm tolerance to BmNPV.
The CRISPR/Cas9 genome editing technology is now widely used in insect studies, but the use of CRISPR can be further increased to improve insect genome engineering. We established a direct mutation at multiple loci in several genes simultaneously used by CRISPR/Cpf1 multiplex genome editing technology to target the BmNPV genome. We constructed a transgenic line that can target the BmNPV ie-1, gp64, and DNApoly genes simultaneously, and hybridized this line with an FnCpf1 transgenic line to obtain an FnCpf1 × gNPVM binary hybrid expression system and to activate the FnCpf1 gene editing system. We showed that the multiple gene editing system introduced deletions, mutations, and insertions at three target sites, and that it did not affect the economic traits of transgenic silkworm lines. The antiviral response of multiplexed genome editing lines increased significantly, and viral gene transcription and replication were significantly affected in the transgenic silkworm lines. This study provides innovative resistance materials for silkworm breeding and also provides a simplified platform for efficient insect multi genome engineering and genetic operation.
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Simple and Efficient Transformation and Gene Editing of Marchantia polymorpha Spores.
Marchantia polymorpha (Marchantia) has become a model species for liverwort studies, owing to its rapid growth in vitro, ease of propagation, simple genetics, small genome, haploid-dominant life cycle, and because it is amenable to genetic transformation. Efficient transformation provides a foundation for many molecular and genetic analyses. The protocol described here is a simple and robust procedure for transforming Marchantia for a variety of applications, including gene overexpression and CRISPR genome editing. This simplified Agrobacterium tumefaciens-mediated transformation protocol targets spores, using common Agrobacterium strains GV3101 or EHA105, and overcomes challenges experienced in other methods. Spores are sterilized and distributed over sterile filter papers, which effectively retain spores and regenerating spores (known as sporelings). This approach enables the sporelings to be transferred to different agar growth media at different stages of transformation. A critical feature is preculturing the spores with acetosyringone (AS) prior to co-cultivation with Agrobacterium. This treatment profoundly enhances the transformation rate, particularly for Agrobacterium strain GV3101. GV3101 is preferred for its rapid growth rate, simple transformation, and lack of a recombinase (recA), stabilizing plasmids. The protocol is suitable for transforming Marchantia with constructs for CRISPR gene editing. Editing efficiency can be increased by introducing a heat-shock treatment during the transformation procedure, which increases the proportion of plants with larger edited sectors, facilitating mutant identification and propagation. Constructs and strategies for both overexpression and multiplex genome editing with sgRNA arrays using new and existing vectors are described. Using this spore transformation protocol for CRISPR gene editing, we routinely achieve 60% to 70% mutation rates, significantly reducing the effort required to generate and isolate mutants for functional analyses. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Agrobacterium-mediated transformation of Marchantia polymorpha spores Support Protocol: Induction of spores APPENDIX 1: Overexpression Vectors APPENDIX 2: Multiplex CRISPR using tRNA arrays.
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CRISPR-Cas9: A cornerstone for the evolution of precision medicine.
Modern genetic therapy incorporates genomic testing and genome editing. It is the finest approach for precision medicine. Genome editing is a state-of-the-art technology to manipulate gene expression thus generating a particular genotype. It encompasses multiple programmable nuclease-based approaches leading to genetic changes. Not surprisingly, this method triggered internationally a wide array of controversies in the scientific community and in the public since it transforms the human genome. Given its importance, the pace of this technology is exceptionally fast. In this report, we introduce one aspect of genome editing, the CRISPR/Cas9 system, highlight its potential to correct genetic mutations and explore its utility in clinical setting. Our goal is to enlighten health care providers about genome editing and incite them to take part of this vital debate.
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CRISPR/Cas9-Mediated Knockout of the White Gene in Agasicles hygrophila.
Agasicles hygrophila is the most effective natural enemy for the control of the invasive weed Alternanthera philoxeroides (Mart.) Griseb. However, research on the gene function and potential genetic improvement of A. hygrophila is limited due to a lack of effective genetic tools. In this study, we employed the A. hygrophila white (AhW) gene as a target gene to develop a CRISPR/Cas9-based gene editing method applicable to A. hygrophila. We showed that injection of Cas9/sgRNA ribonucleoprotein complexes (RNPs) of the AhW gene into pre-blastoderm eggs induced genetic insertion and deletion mutations, leading to white eyes. Our results demonstrate that CRISPR/Cas9-mediated gene editing is possible in A. hygrophila, offering a valuable tool for studies of functional genomics and genetic improvement of A. hygrophila, which could potentially lead to more effective control of invasive weeds through the development of improved strains of this biocontrol agent. In addition, the white-eyed mutant strain we developed could potentially be useful for other transgenic research studies on this species.
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Walk the Line: Debating a Germline Editing Moratorium.
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From gene editing to tumor eradication: The CRISPR revolution in cancer therapy.
Cancer continues to be a significant worldwide health concern, characterized by high rates of occurrence and death. Unfortunately, existing treatments frequently fall short of delivering satisfying therapeutic outcomes. Immunotherapy has ushered in a new era in the treatment of solid tumors, yet its effectiveness is still constrained and comes with unwanted side effects. The advancement of cutting-edge technology, propelled by gene analysis and manipulation at the molecular scale, shows potential for enhancing these therapies. The advent of genome editing technologies, including CRISPR-Cas9, can greatly augment the efficacy of cancer immunotherapy. This review explores the mechanism of CRISPR-Cas9-mediated genome editing and its wide range of tools. The study focuses on analyzing the effects of CRISPR-induced double-strand breaks (DSBs) on cancer immunotherapy, specifically by gene knockdown or knockin. In addition, the study emphasizes the utilization of CRISPR-Cas9-based genome-wide screening to identify targets, the potential of spatial CRISPR genomics, and the extensive applications and difficulties of CRISPR-Cas9 in fundamental research, translational medicine, and clinical environments.
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CRISPR Technology in Cancer Diagnosis and Treatment: Opportunities and Challenges.
A novel gene editing tool, the Cas system, associated with the CRISPR system, is emerging as a potential method for genome modification. This simple method, based on the adaptive immune defense system of prokaryotes, has been developed and used in human cancer research. These technologies have tremendous therapeutic potential, especially in gene therapy, where a patient-specific mutation is genetically corrected to cure diseases that cannot be cured with conventional treatments. However, translating CRISPR/Cas9 into the clinic will be challenging, as we still need to improve the efficiency, specificity, and application of the technology. In this review, we will explain how CRISPR-Cas9 technology can treat cancer at the molecular level, focusing on ordination and the epigenome. We will also focus on the promise and shortcomings of this system to ensure its application in the treatment and prevention of cancer.
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Primate Organoids and Gene-Editing Technologies toward Next-Generation Biomedical Research.
The improved ability to organize pluripotent stem cells (PSCs) into 3D structures in vitro has shed light on organoid technology to recapitulate organs and tumors in vivo. Advances in gene-editing technologies, particularly CRISPR-mediated techniques, offer tremendous potential in facilitating organoid research, including the study of development, disease modeling, and personalized medicine. This review discusses how the combination of two novel technologies - organoids and gene editing - not only contributes to revealing molecular events taking place during development and tumorigenesis but also has implications for biobanking, precision medicine, and other diverse biomedical applications.
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The case for germline gene correction: state of the science.
Germline gene editing refers to altering the DNA in the reproductive cells (gametes or embryos). Germline gene editing experiments in human embryos have primarily focused on correcting genetic mutations linked to inherited diseases. This technology has the potential to prevent genetic disease before birth and in future generations. Advances in CRISPR-Cas9 and other gene-editing tools have accelerated scientific progress, raising both promise and safety and ethical concerns. A translational pathway for human heritable genome editing will require an approach that integrates scientific validation, ethical oversight, regulatory frameworks, and public engagement.
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