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The way to true plant genome editing.
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CRISPR/Cas9 Genome Editing in Oncology: Mechanisms, Therapeutic Platforms and Translational Challenges.
The CRISPR/Cas9 genome editing technology has had a significant impact on cancer research and therapeutic development, providing unprecedented precision in manipulating cancer-associated genes. Although this review focuses on Cas9, we situate it within the broader CRISPR landscape that includes DNA-targeting effectors (Cas9/Cas12), RNA-targeting systems such as Cas13, and type III systems with dual DNA and RNA activity, modalities that expand both experimental and therapeutic possibilities. This comprehensive review examines the current applications of CRISPR/Cas9 in oncology, including its mechanisms and the challenges associated with its clinical translation. Knockout, interference, and activation CRISPR screening platforms have transformed functional genomics by systematically interrogating gene function, identifying therapeutic vulnerabilities, and clarifying resistance mechanisms across diverse cancer phenotypes. This technology has also reshaped cancer modeling, enabling precise recapitulation of disease-relevant mutations from engineered cell lines to patient-derived xenografts that capture tumor heterogeneity and microenvironmental interactions. Notably, the integration of CRISPR/Cas9 with CAR-T therapy has enabled multiplex editing to eliminate alloreactivity, overcome checkpoint-mediated exhaustion, and engineer universal CAR-T cells. Emerging in vivo strategies that directly generate or reprogram CAR-T cells in patients via targeted viral and nonviral delivery underscore accelerating translational momentum. However, significant challenges, including off-target mutagenesis, delivery barriers, p53-mediated selective pressure favoring potentially oncogenic populations, and Cas9 immunogenicity, continue to hinder clinical translation. These limitations necessitate high-fidelity nucleases, optimized guide designs, and improved delivery systems. The future of CRISPR/Cas9 in cancer therapy will depend on technological innovation, comprehensive safety frameworks, and rigorous clinical evaluation as next-generation editing modalities advance toward transformative precision oncology.
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Ethical aspects associated with genome alteration techniques applied to animal reproduction research.
The revolution in biology triggered by the different genome-editing tools has of course arrived to the research field of animal reproduction. Yeast meganucleases, zinc-finger nucleases, TALEN and, particularly, the several generations of CRISPR tools have landed in animal reproduction thereby providing novel strategies to optimize or modify some of the features and capabilities of the recipient animals. All these genome-editing proposals and activities are associated with ethical considerations regarding how those planned genome alterations might affect important animal welfare issues. The ethical dimension of all these genome editing must be seriously considered. Hence, all ethical aspects bound to any given genome-edited allele in animals should be discussed in order to ensure that we are maximizing benefits and reducing any potential risk or negative considerations of these modifications. In this review, I will summarize some of the experiments reported aiming to investigate or improve animal reproduction and I will address the ethics issues that should also be considered.
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Beginning of life ethics at the dawn of a new era of genome editing: are bioethical precepts and fast-evolving biotechnologies irreconcilable?
The amazing and almost unimaginable advances that have unfolded over the past decades in biotechnologies (heritable germline editing in particular) have brought bioethical issues to the forefront, sparking public debate and increasing attention worldwide. Such mind-blowing progress has already resulted in major improvement and enhancements for humans, and holds the potential for even more. Technology and bioengineering have begun to take over in the life sciences industry. Man's capacity to genetically engineer the biological world is nothing short of mind-boggling in its current magnitude, and may even evolve, in a not too distant future, into attempts to fuse man and machine into a cohesive bioengineered entity; a "super human being", endowed with enhanced cognitive and physical capabilities and impervious to disease, may be not too far down the road. That will not come without caveats, however. In fact, scientific advancements at such an accelerated pace have already negatively affected our cultural, ethical, and legal values and our ability to harness the opportunities and face the dangers posed by such developments. As a matter of fact, science seems to consistently outpace public morals, ethics and policymaking, which calls for a high degree of caution and common answers.
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The new CRISPR-Cas system: RNA-guided genome engineering to efficiently produce any desired genetic alteration in animals.
The CRISPR-Cas system is the newest targeted nuclease for genome engineering. In less than 1 year, the ease, robustness and efficiency of this method have facilitated an immense range of genetic modifications in most model organisms. Full and conditional gene knock-outs, knock-ins, large chromosomal deletions and subtle mutations can be obtained using combinations of clustered regularly interspaced short palindromic repeats (CRISPRs) and DNA donors. In addition, with CRISPR-Cas compounds, multiple genetic modifications can be introduced seamlessly in a single step. CRISPR-Cas not only brings genome engineering capacities to species such as rodents and livestock in which the existing toolbox was already large, but has also enabled precise genetic engineering of organisms with difficult-to-edit genomes such as zebrafish, and of technically challenging species such as non-human primates. The CRISPR-Cas system allows generation of targeted mutations in mice, even in laboratories with limited or no access to the complex, time-consuming standard technology using mouse embryonic stem cells. Here we summarize the distinct applications of CRISPR-Cas technology for obtaining a variety of genetic modifications in different model organisms, underlining their advantages and limitations relative to other genome editing nucleases. We will guide the reader through the many publications that have seen the light in the first year of CRISPR-Cas technology.
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An efficient genetic transformation and gene editing system mediated by Agrobacterium rhizogenes for Liriodendron hybrid and its application.
An efficient genetic transformation system is imperative for advancing gene functional studies and molecular breeding in horticultural tree species. Traditional Agrobacterium tumefaciens-mediated methods have encountered significant technical challenges when applied to tree species, particularly Liriodendron hybrids. The high costs, and extended transformation cycles associated with this method have substantially limited its utility in gene functional analysis and breeding applications. In response to these challenges, this study presents the development of a streamlined, rapid, and efficient Agrobacterium rhizogenes-mediated transformation system tailored specifically for Liriodendron hybrids, an important ornamental and timber tree species. Among the three Agrobacterium rhizogenes strains (K599, MSU440, and C58C1) evaluated in this study, K599 demonstrated the highest transformation efficiency, reaching 46.09 % for inducing hairy roots from apical bud incisions. Further optimization of the system revealed that 2 days and two-month-old seedlings were the most suitable co-culture duration and explants, yielding a peak transformation efficiency of 60.38 %, 53.12 %, respectively. The applicability of this transformation system was validated across various Liriodendron hybrids genotypes and Liriodendron tulipifera, with transformation efficiencies ranging from 15.47 % to 60.63 %. Additionally, the system was effectively employed for subcellular localization analysis, which confirmed that the aquaporin (AQP) protein. LhAQP1 is localized in the plasma membrane and exhibits enrichment in the vascular tissues of the hairy roots. Notably, this study marks the first application of the Agrobacterium rhizogenes-mediated system for CRISPR/Cas9-mediated gene editing in Liriodendron hybrids, successfully achieving targeted mutagenesis of the LhAQP1 gene and establishing the feasibility of gene editing within this species. In addition, the hairy root-based transformation system was employed to investigate the functional role of LhAQP1 in the improved Liriodendron variety 'Nanlin-Jinsen E1' by comparing wild-type, LhAQP1-overexpressing and gene-edited lines generated via the CRISPR/Cas9. LhAQP1-overexpression significantly promoted plant growth and drought tolerance, whereas pYLCRISPR-LhAQP1 increases dehydration sensitivity, underscoring the essential role of LhAQP1 in water stress adaptation. Optimized transformation platform represents a crucial advancement for functional genomics and molecular breeding efforts in woody plants.
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Establishment and application of a silkworm CRISPR/Cas9 tool for conditionally manipulating gene disruption in the epidermis.
Spatial or temporal specific gene knockout system is a valuable tool for studying the molecular mechanisms underlying developmental processes. The integument is essential for insect fitness and survival, but tools for dissecting function of genes in this tissue are lacking. In this study, we firstly identified an epidermis specifically expressed gene of the domesticated silkworm, BmCPG25, by comparative transcriptomic analysis. Furthermore, a transgenic silkworm expressing the RNA dependent CRISPR-Cas9 protein driven by the regulatory region of the BmCPG25 was established. Immunochemistry analysis showed the endonuclease was specifically expressed in the nuclear of epidermal cells. We also validated the efficiency of this system by disrupting the function of an epidermis specifically expressed cuticular protein gene (Cpr21) and a ubiquitously expressed cuticular gene (Cph18), respectively. In summary, we successfully constructed a conditional knockout toolkit to manipulate the gene editing in epidermal cells, which provides a valuable approach to study the molecular mechanism of integument development.
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Cas9-PALB2 fusion protein enhances CRISPR/Cas9 mediated gene knock-in efficiency.
Over the past decade, CRISPR-based technologies have revolutionized our capacity to manipulate genomes, thereby reshaping the landscape of functional genomics research. Among the CRISPR toolkit, CRISPR/Cas9-mediated homology-directed repair (HDR) enables precise genome editing with predefined mutations, rendering it an indispensable tool for gene functional analysis, disease model construction, and the development of gene therapy strategies. Nevertheless, despite the robust efficiency of CRISPR/Cas9 in mediating gene knockouts, HDR-dependent gene knock-in (KI) remains a major bottleneck due to its inherently low efficiency. Herein, we report that the co-expression of PALB2 with the CRISPR/Cas9 nuclease could trigger an enhanced HDR effect. Specifically, the fusion of Cas9 with PALB2 elevated KI efficiency by approximately 1.7-fold in human HEK293T cells. Furthermore, this Cas9-PALB2 fusion strategy exhibited cross-cell-type efficacy, demonstrating its broad applicability beyond a single cell line. Notably, the combined application of the Cas9-PALB2 fusion system and Nocodazole further boosted KI efficiency to a remarkable 25.5%. Collectively, these findings establish the Cas9-PALB2 fusion as a highly potent and versatile strategy to augment HDR-mediated KI efficiency, highlighting its substantial potential for widespread utilization in applications that demand high-fidelity genome editing.
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Targeted Genome Editing of Virulent Pseudomonas Phages Using CRISPR-Cas3.
The vast number of unknown phage-encoded ORFan genes and limited insights into the genome organization of phages illustrate the need for efficient genome engineering tools to study bacteriophage genes in their natural context. In addition, there is an application-driven desire to alter phage properties, which is hampered by time constraints for phage genome engineering in the bacterial host. We here describe an optimized CRISPR-Cas3 system in Pseudomonas for straightforward editing of the genome of virulent bacteriophages. The two-vector system combines a broad host range CRISPR-Cas3 targeting plasmid with a SEVA plasmid for homologous directed repair, which enables the creation of clean deletions, insertions, or substitutions in the phage genome within a week. After creating the two plasmids separately, a co-transformation to P. aeruginosa cells is performed. A subsequent infection with the targeted phage allows the CRISPR-Cas3 system to cut the DNA specifically and facilitate or select for homologous recombination. This system has also been successfully applied for P. aeruginosa and Pseudomonas putida genome engineering. The method is straightforward, efficient, and universal, enabling to extrapolate the system to other phage-host pairs.
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High-efficiency CRISPR/Cas-based editing of Phalaenopsis orchid MADS genes.
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