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Removal of extra sequences with I-SceI in combination with CRISPR/Cas9 technique for precise gene editing in Drosophila.
The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies and has been adopted for many organisms, including Drosophila. Previously, an efficient two-step strategy was developed to engineer the fly genome by combining CRISPR/Cas9 with recombinase-mediated cassette exchange (RMCE). This strategy allows the introduction of designed mutations into a gene of interest in vivo. However, the loxP or frt site remains in the edited locus. Here, we propose a modification of this approach for rapid and efficient seamless genome editing with CRISPR/Cas9 and site-specific recombinase-mediated integration (SSRMI) combined with recombination between homologous sequences induced by the rare-cutting endonuclease I-SceI. The induced homological recombination leads to the removal of the remaining extraneous sequences from the target locus.
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Next-generation CAR-T cell therapy against cancer: precision engineering, programmable immunity, and emerging clinical frontiers.
While chimeric antigen receptor T-cell (CAR-T) therapy has revolutionized the treatment of hematological malignancies, there are significant hurdles to overcome, such as antigen escape, T-cell exhaustion, limited persistence in the body, severe toxicities, manufacturing complexity, high costs, and low effectiveness in the treatment of solid tumors. There are many reviews describing novel CAR-T technologies, the evidence strength, clinical maturity and unmet translational risks of each are largely included in those reviews. This review highlights the ways in which next-generation engineering increases the safety, efficacy and programmability of CAR-T. The primary and authoritative clinical, regulatory and preclinical evidence on recent advances from 2022 to 2026 was carefully evaluated, including the quality and quality control of the evidence for each strategy. New innovations are Armored CARs, dual-target and logic-gated/synNotch systems, universal and switchable platforms, CRISPR/Cas9 and base/prime editing, allogeneic CAR-Ts, non-viral manufacturing, and lipid-nanoparticles in vivo CAR-T generation. Additionally, the engineering and combination approaches for the treatment of solid tumors with checkpoint inhibitors, radiotherapy, oncolytic viruses, nanotechnology, and artificial intelligence are discussed. An evidence-to-maturity framework separates the clinically proven methods from the emerging technologies and highlights some of the critical translation hurdles related to safety, immunogenicity, durability, manufacturing and scale. Precision Immunotherapy is becoming more and more possible with the next generation CAR-T engineering, however the validation and translational optimization of these cells is dominant to their eventual clinical use.
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Principles of gene editing techniques and applications in animal husbandry.
Gene editing techniques were developed chronologically, which include zinc finger nuclease, transcription activator-like effector nuclease and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas 9). In this review, the working principles of these techniques were first introduced, their advantages and disadvantages were then discussed, their application in animal husbandry were elaborated, and finally human concerns about gene editing were presented. Compared to the two former techniques, the third-generation gene editing technique CRISPR/Cas9 has higher targeting efficiency and accuracy, less off-target effect, lower cytotoxicity and lower costs for being easier for vector design and manipulation. Although some people may concern about social or ethical issues, the benefits of gene editing certainly overweigh its demerits. The three gene editing techniques have been successfully used to improve the production and quality of livestock products, animal fertility, resistance to diseases, and welfare in animal husbandry. With legislation and the development of gene editing technology per se, it anticipatable that gene editing will have a broader utilization and make our lives happier.
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Gene Editing in Humans: Towards a Global and Inclusive Debate for Responsible Research.
In December 2016, the Opinion Group of the Bioethics and Law Observatory (OBD) of the University of Barcelona launched a Declaration on Bioethics and Gene Editing in Humans analyzing the use of genome editing techniques and their social, ethical, and legal implications through a multidisciplinary approach. It focuses on CRISPR/Cas9, a genome modification technique that enables researchers to edit specific sections of the DNA sequence of humans and other living beings. This technique has generated expectations and worries that deserve an interdisciplinary analysis and an informed social debate. The research work developed by the OBD presents a set of recommendations addressed to different stakeholders and aims at being a tool to learn more about CRISPR/Cas9 while finding an appropriate ethical and legal framework for this new technology. This article gathers and compares reports that have been published in Europe and the USA since the OBD Declaration. It aims at being a tool to foster a global and interdisciplinary discussion of this new genome editing technology.
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Multiplex genome editing of microorganisms using CRISPR-Cas.
Microbial production of chemical compounds often requires highly engineered microbial cell factories. During the last years, CRISPR-Cas nucleases have been repurposed as powerful tools for genome editing. Here, we briefly review the most frequently used CRISPR-Cas tools and describe some of their applications. We describe the progress made with respect to CRISPR-based multiplex genome editing of industrial bacteria and eukaryotic microorganisms. We also review the state of the art in terms of gene expression regulation using CRISPRi and CRISPRa. Finally, we summarize the pillars for efficient multiplexed genome editing and present our view on future developments and applications of CRISPR-Cas tools for multiplex genome editing.
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CRISPR-Cas9 system: A genome-editing tool with endless possibilities.
The discovery of CRISPR: Cas9 and its application as a powerful gene-editing tool has transformed the world of basic and applied science, especially the molecular biology dome. Also, the smooth, quick, flexible, and very efficient nature of this technology has enabled the biologists to alter the genome of prokaryotes to complex eukaryotic systems, including plants and animals. Using CRISPR and associated tools, investigation, control, and modification of significant biological events have been more accessible than before. These biological scissors are now being used to accelerate breeding programs of crop and livestock, engineer new antimicrobials, and control disease-carrying pathogens. However, like other techniques, these cutters emerged as a double-edged sword and put several challenges to the scientific society. Here in this review article, we summarized the beneficial application of the CRISPR: Cas9 system and unsafe perception to the society if handled carelessly. We also discussed the limitations and ethical issues related to CRISPR: Cas9 technology.
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A Photolabile Semiconducting Polymer Nanotransducer for Near-Infrared Regulation of CRISPR/Cas9 Gene Editing.
Noninvasive regulation of CRISPR/Cas9 gene editing is conducive to understanding of gene function and development of gene therapy; however, it remains challenging. Herein, a photolabile semiconducting polymer nanotransducer (pSPN) is synthesized to act as the gene vector to deliver CRISPR/Cas9 plasmids into cells and also as the photoregulator to remotely activate gene editing. pSPN comprises a 1 O2 -generating backbone grafted with polyethylenimine brushes through 1 O2 -cleavable linkers. NIR photoirradiation spontaneously triggers the cleavage of gene vectors from pSPN, resulting in the release of CRISPR/Cas9 plasmids and subsequently initiating gene editing. This system affords 15- and 1.8-fold enhancement in repaired gene expression relative to the nonirradiated controls in living cells and mice, respectively. As this approach does not require any specific modifications on biomolecular components, pSPN represents the first generic nanotransducer for in vivo regulation of CRISPR/Cas9 gene editing.
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Therapeutic applications of gene editing using CRISPR-Cas9 in the posterior segment: review of the literature.
CRISPR-Cas gene editing has become increasingly relevant in the treatment of several ophthalmic diseases. Its ability to make precise modifications at the DNA or RNA level has enabled targeted approaches for specific mutations involved in conditions such as Leber congenital amaurosis type 10 (LCA10), certain forms of retinitis pigmentosa, and age-related macular degeneration. This article provides an organized overview of the biological basis of CRISPR-Cas technology and highlights key advances from preclinical studies and early clinical trials. Technical limitations and ongoing safety challenges are also discussed. Programs such as EDIT-101, EDIT-103, and HG202 stand out as important milestones in the evolution of ocular gene editing.
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Application of the gene editing tool, CRISPR-Cas9, for treating neurodegenerative diseases.
Increased accumulation of transcribed protein from the damaged DNA and reduced DNA repair capability contributes to numerous neurological diseases for which effective treatments are lacking. Gene editing techniques provide new hope for replacing defective genes and DNA associated with neurological diseases. With advancements in using such editing tools as zinc finger nucleases (ZFNs), meganucleases, and transcription activator-like effector nucleases (TALENs), etc., scientists are able to design DNA-binding proteins, which can make precise double-strand breaks (DSBs) at the target DNA. Recent developments with the CRISPR-Cas9 gene-editing technology has proven to be more precise and efficient when compared to most other gene-editing techniques. Two methods, non-homologous end joining (NHEJ) and homology-direct repair (HDR), are used in CRISPR-Cas9 system to efficiently excise the defective genes and incorporate exogenous DNA at the target site. In this review article, we provide an overview of the CRISPR-Cas9 methodology, including its molecular mechanism, with a focus on how in this gene-editing tool can be used to counteract certain genetic defects associated with neurological diseases. Detailed understanding of this new tool could help researchers design specific gene editing strategies to repair genetic disorders in selective neurological diseases.
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Research Progress and Application of Miniature CRISPR-Cas12 System in Gene Editing.
CRISPR-Cas system, a natural acquired immune system in prokaryotes that defends against exogenous DNA invasion because of its simple structure and easy operation, has been widely used in many research fields such as synthetic biology, crop genetics and breeding, precision medicine, and so on. The miniature CRISPR-Cas12 system has been an emerging genome editing tool in recent years. Compared to the commonly used CRISPR-Cas9 and CRISPR-Cas12a, the miniature CRISPR-Cas12 system has unique advantages, such as rich PAM sites, higher specificity, smaller volume, and cytotoxicity. However, the application of miniature Cas12 proteins and the methods to improve its editing efficiency have not been systematically summarized. In this review, we introduce the classification of CRISPR-Cas system and summarize the structural characteristics of type V CRISPR-Cas system and the cleavage mechanism of five miniature Cas12 proteins. The application of a miniature CRISPR-Cas12 system in the gene editing of animals, plants, and microorganisms is summarized, and the strategies to improve the editing efficiency of the miniature CRISPR-Cas12 system are discussed, aiming to provide reference for further understanding the functional mechanism and engineering modification of the miniature CRISPR-Cas12 system.
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