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Effect of gamma-ray exposure on the genome-editing efficiency of improved genome-editing via oviductal nucleic acids delivery (i-GONAD).
DNA double-strand breaks (DSBs) are among the most hazardous cellular damages, potentially leading to cell death or oncogenesis if unrepaired. Genome editing methods, such as the CRISPR/Cas9 system, induce DSBs and utilize these repair pathways for gene knockout and knock-in. Although ionizing radiation also induces DSBs, it is not clear whether the efficiency of genome editing is affected by ionizing radiation. This study investigated the impact of gamma-ray exposure on the genome editing efficiency of the improved genome editing via oviductal nucleic acid delivery (i-GONAD) method. Gamma-rays were exposed to pregnant mice receiving i-GONAD targeting the Hr gene, whose mutation causes hair loss in mice. The exposure on the fertilization day (Day 0) decreased natural delivery rates and litter sizes, with notable effects at 0.3 Gy or higher. Although the proportions of hairless offspring obtained by i-GONAD differed greatly between single-guide RNAs (sgRNAs) used, total mutation rates, including hairless, mosaic, and indel, were equivalent. Gamma-ray exposure on Day 0 and the day after fertilization (Day 1) similarly and almost dose-dependently enhanced the genome editing efficiency evaluated by the total mutation rate. This study suggests the improvement of genome editing efficiency by gamma-ray exposure, at least in i-GONAD method, potentially facilitating the creation of diverse experimental animal models.
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Application of CHyMErA Cas9-Cas12a combinatorial genome-editing platform for genetic interaction mapping and gene fragment deletion screening.
CRISPR-based forward genetic screening represents a powerful approach for the systematic characterization of gene function. Recent efforts have been directed toward establishing CRISPR-based tools for the programmable delivery of combinatorial genetic perturbations, most of which are mediated by a single nuclease and the expression of structurally identical guide backbones from two promoters. In contrast, we have developed CHyMErA (Cas hybrid for multiplexed editing and screening applications), which is based on the co-expression of Cas9 and Cas12a nucleases in conjunction with a hybrid guide RNA (hgRNA) engineered by the fusion of Cas9 and Cas12a guides and expressed from a single U6 promoter. CHyMErA is suitable for the high-throughput deletion of genetic segments including the excision of individual exons. Furthermore, CHyMErA enables the concomitant targeting of two or more genes and can thus be used for the systematic mapping of genetic interactions in mammalian cells. CHyMErA can also be applied for the perturbation of paralogous gene pairs, thereby allowing the capturing of phenotypic roles that would otherwise be masked because of genetic redundancy. Here, we provide instructions for the cloning of hgRNA screening libraries and individual hgRNA constructs and offer guidelines for designing and performing combinatorial pooled genetic screens using CHyMErA. Starting with the generation of Cas9- and Cas12a-expressing cell lines, CHyMErA screening can be implemented within 15-20 weeks.
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Efficient engineering of human and mouse primary cells using peptide-assisted genome editing.
Simple, efficient and well-tolerated delivery of CRISPR genome editing systems into primary cells remains a major challenge. Here we describe an engineered Peptide-Assisted Genome Editing (PAGE) CRISPR-Cas system for rapid and robust editing of primary cells with minimal toxicity. The PAGE system requires only a 30-min incubation with a cell-penetrating Cas9 or Cas12a and a cell-penetrating endosomal escape peptide to achieve robust single and multiplex genome editing. Unlike electroporation-based methods, PAGE gene editing has low cellular toxicity and shows no significant transcriptional perturbation. We demonstrate rapid and efficient editing of primary cells, including human and mouse T cells, as well as human hematopoietic progenitor cells, with editing efficiencies upwards of 98%. PAGE provides a broadly generalizable platform for next-generation genome engineering in primary cells.
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Integration of xeno-free single-cell cloning in CRISPR-mediated DNA editing of human iPSCs improves homogeneity and methodological efficiency of cellular disease modeling.
The capability to generate induced pluripotent stem cell (iPSC) lines, in tandem with CRISPR-Cas9 DNA editing, offers great promise to understand the underlying genetic mechanisms of human disease. The low efficiency of available methods for homogeneous expansion of singularized CRISPR-transfected iPSCs necessitates the coculture of transfected cells in mixed populations and/or on feeder layers. Consequently, edited cells must be purified using labor-intensive screening and selection, culminating in inefficient editing. Here, we provide a xeno-free method for single-cell cloning of CRISPRed iPSCs achieving a clonal survival of up to 70% within 7-10 days. This is accomplished through improved viability of the transfected cells, paralleled with provision of an enriched environment for the robust establishment and proliferation of singularized iPSC clones. Enhanced cell survival was accompanied by a high transfection efficiency exceeding 97%, and editing efficiencies of 50%-65% for NHEJ and 10% for HDR, indicative of the method's utility in stem cell disease modeling.
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Disrupting off-target Cas9 activity in the liver.
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A Cryoprotectant-Compatible Nanoporous Platform for Stable and Scalable Delivery of Biopharmaceuticals.
CRISPR-Cas9 ribonucleoproteins (RNPs) represent a promising class of biopharmaceuticals for treating genetic and complex diseases. However, their clinical translation is limited by instability during storage and delivery. Lyophilization offers a potential solution, though conventional approaches often compromise structural integrity and bioactivity under non-cryogenic conditions. Here, we have developed a nanostructured delivery platform, designated Nano Banker & Blowball (NB2), which features a blowball-like architecture and tunable nanoscale pores. These pores are designed to protect RNPs and enable controlled release. The freeze-dried formulation (FNB2) integrates optimized cryoprotectants and a surface-engineered nanoparticle design, preserving morphology and function without excessive excipients. FNB2 exhibits rapid rehydration and retains ∼70% of gene editing activity post-lyophilization, enabling robust functional gene editing in vitro and in vivo. It also maintains long-term stability and supports efficient cellular uptake, enabling administration via multiple routes. FNB2 represents a scalable and robust platform for genetic therapeutics, vaccines, and biologics, particularly well-suited for resource-limited and emergency medical applications.
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The path to biotechnological singularity: Current breakthroughs and outlook.
Fueled by rapid advances in gene editing, synthetic biology, artificial intelligence, regenerative medicine, and brain-computer interfaces, biotechnology is approaching a transformative era often referred to as biotechnological singularity. CRISPR-based gene editing has revolutionized genetic engineering, enabling precise modifications for treating hereditary diseases and cancer. Synthetic biology facilitates sustainable biomaterial production and innovative therapeutic applications. Artificial intelligence accelerates drug discovery, enhances diagnostic accuracy, and personalizes treatment through deep learning models. Driven by stem cell research, regenerative medicine offers promising avenues for reversing aging and treating degenerative diseases. Brain-computer interfaces merge human cognition with technology, enabling direct neural control of prosthetics and expanding human-machine interactions. These breakthroughs, however, raise ethical, regulatory, and societal concerns, including equitable access, biosecurity risks, and the implications of human enhancement. The convergence of biological and computational technologies challenges traditional boundaries, necessitating comprehensive governance frameworks. By embracing responsible innovation, society can harness these advancements for transformative health interventions, environmental sustainability, and extended longevity. The realization of biotechnological singularity depends on interdisciplinary collaboration among scientists, policymakers, and the public to ensure that progress aligns with the well-being of humanity and ethical considerations.
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Cas-analyzer: an online tool for assessing genome editing results using NGS data.
UNLABELLED: Genome editing with programmable nucleases has been widely adopted in research and medicine. Next generation sequencing (NGS) platforms are now widely used for measuring the frequencies of mutations induced by CRISPR-Cas9 and other programmable nucleases. Here, we present an online tool, Cas-Analyzer, a JavaScript-based implementation for NGS data analysis. Because Cas-Analyzer is completely used at a client-side web browser on-the-fly, there is no need to upload very large NGS datasets to a server, a time-consuming step in genome editing analysis. Currently, Cas-Analyzer supports various programmable nucleases, including single nucleases and paired nucleases. AVAILABILITY AND IMPLEMENTATION: Free access at http://www.rgenome.net/cas-analyzer/ CONTACT: sangsubae@hanyang.ac.kr or jskim01@snu.ac.krSupplementary information: Supplementary data are available at Bioinformatics online.
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Generation of Knockout Human Primary Keratinocytes by CRISPR/Cas9.
The culture of epidermal human primary keratinocytes (HPKs) represents a well-established model in biological and dermatological research. In addition, HPKs are used in three-dimensional organotypic cultures (OTCs), and gene therapeutic approaches have been reported for the treatment of patients suffering from epidermolysis bullosa, a severe blistering disease that can result in postnatal lethality. Therefore, there is a strong need for the development of techniques for the stable and specific genetic manipulation of HPKs, for example, by genome editing via the CRISPR/Cas9 approach. However, the main disadvantage of working with HPKs is the fact that these cells are prone to terminal differentiation and proliferate only for few passages in monoculture. As it is well known that the co-culture of HPKs with fibroblasts strongly increases the lifetime of the epidermal cells, we developed a protocol for the stable modification of HPKs by CRISPR/Cas9 via lentiviral transduction in the presence of 3T3-J2 fibroblasts as feeder cells. Selection of transduced HPKs is achieved with antibiotics in co-culture with antibiotic-resistant feeder cells. Modified HPKs generated by our protocol have the potential to generate epidermis-like structures in OTCs.
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Advances in CRISPR-Cas systems for blood cancer.
CRISPR-Cas systems have revolutionised precision medicine by enabling personalised treatments tailored to an individual's genetic profile. Various CRISPR technologies have been developed to target specific disease-causing genes in blood cancers, and some have advanced to clinical trials. Although some studies have explored the in vivo applications of CRISPR-Cas systems, several challenges continue to impede their widespread use. Furthermore, CRISPR-Cas technology has shown promise in improving the response of immunotherapies to blood cancers. The emergence of CAR-T cell therapy has shown considerable success in the targeting and correcting of disease-causing genes in blood cancers. Despite the promising potential of CRISPR-Cas in the treatment of blood cancers, issues related to safety, ethics, and regulatory approval remain significant hurdles. This comprehensive review highlights the transformative potential of CRISPR-Cas technology to revolutionise blood cancer therapy.
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