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Advancing Mesenchymal Stem Cell Therapy with CRISPR/Cas9 for Clinical Trial Studies.
Currently, regenerative medicine and cellular-based therapy have been in the center of attention worldwide in advanced medical technology. Mesenchymal stem cell (MSC) as a suitable stem cell source for cell-based therapy has been shown to be safe and effective in multiple clinical trial studies (CTSs) of several diseases. Despite the advantages, MSC needs more investigation to enhance its therapeutic application. The CRISPR/Cas system is a novel technique for editing of genes that is being explored as a means to improve MSCs therapeutic usage. In this study, we review the recent studies that explore CRISPR potency in gene engineering of MSCs, which have great relevance in MSC-based therapies. However, CRISPR/Cas technology make possible specific targeting of loci in target genes, but next-generation MSC-based therapies to achieve extensive clinical application need dedicated efforts.
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Biologically produced and metal-organic framework delivered dual-cut CRISPR/Cas9 system for efficient gene editing and sensitized cancer therapy.
Manipulation of the lactate metabolism is an efficient way for cancer treatment given its involvement in cancer development, metastasis, and immune escape. However, most of the inhibitors of lactate transport carriers suffer from poor specificity. Herein, we use the CRISPR/Cas9 system to precisely downregulate the monocarboxylate carrier 1 (MCT1) expression. To avoid the self-repairing during the gene editing process, a dual-Cas9 ribonucleoproteins (duRNPs) system is generated using the biological fermentation method and delivered into cells by the zeolitic imidazolate framework-8 (ZIF-8) nanoparticles, enabling precise removal of a specific DNA fragment from the genome. For efficient cancer therapy, a specific glucose transporter 1 inhibitor (BAY-876) is co-delivered with the duRNPs, forming BAY/duRNPs@ZIF-8 nanoparticle. ZIF-8 nanoparticles can deliver the duRNPs into cells within 1 h, which efficiently downregulates the MCT1 expression, and prohibits lactate influx. Through simultaneous inhibition of the lactate and glucose influx, BAY/duRNPs@ZIF-8 prohibits ATP generation, arrests cell cycle, inhibits cell proliferation, and finally induces cellular apoptosis both in vitro and in vivo. Consequently, we demonstrate that the biologically produced duRNPs delivered into cells by the nonviral ZIF-8 carrier have expanded the CRISPR/Cas gene editing toolbox and elevated the gene editing efficiency, which will promote biological studies and clinical applications. STATEMENT OF SIGNIFICANCE: The CRISPR/Cas9 system, widely used as an efficient gene editing tool, faces a challenge due to cells' ability to self-repair. To address this issue, a strategy involving dual-cutting of the genome DNA has been designed and implemented. This strategy utilizes biologically produced dual-ribonucleoproteins delivered by a metal-organic framework. The effectiveness of this dual-cut CRISPR-Cas9 system has been demonstrated through a therapeutic approach targeting the simultaneous inhibition of lactate and glucose influx in cancer cells. The utilization of the dual-cut gene editing strategy has provided valuable insights into gene editing and expanded the toolbox of the CRISPR/Cas-based gene editing system. It has the potential to enable more efficient and precise manipulation of specific protein expression in the future.
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CRISPR/Cas9-Mediated Targeted Mutagenesis in Wheat Doubled Haploids.
CRISPR/Cas9-based genome editing technology has the potential to revolutionize agriculture, but many plant species and/or genotypes are recalcitrant to conventional transformation methods. Additionally, the long generation time of crop plants poses a significant obstacle to effective application of gene editing technology, as it takes a long time to produce modified homozygous genotypes. The haploid single-celled microspores are an attractive target for gene editing experiments, as they enable generation of homozygous doubled haploid mutants in one generation. Here, we describe optimized methods for genome editing of haploid wheat microspores and production of doubled haploid plants by microspore culture.
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An optimised lipid nanoparticle platform enables efficient CRISPR/Cas9 genome editing in hard-to-transfect cells.
CRISPR/Cas9 is a powerful tool for genome editing and functional gene studies, but its therapeutic potential is often hampered by inefficient transfection, particularly in hard-to-modify cell types. In this study, we developed and optimised a lipid nanoparticles (LNPs) platform that enhances CRISPR/Cas9-mediated genome editing across diverse cell types, including those that are difficult to modify using commercially available lipid-based delivery agents. Our engineered LNPs exhibit consistent particle size below 100 nm, low polydispersity and high encapsulation efficiency. Using this platform, GFP knockout in HEK-293 cells reached 78.7%, and maintained consistent efficiency after lyophilisation and reconstitution. Knockout of the LCN2 gene in MDA-MB-231 cells resulted in a 90.8% reduction in mRNA expression, outperforming the 51.1% reduction achieved using CRISPRMAX Lipofectamine, and functional assays confirmed that LCN2 disruption significantly inhibited cell proliferation and migration. Co-delivery of CRISPR/Cas9 and a GFP HDR template enabled precise knock-in, achieving >20% efficiency in HEK-293 cells and >8% in MSCs and DC2.4 cells, significantly outperforming Lipofectamine 3000 transfection reagent. Given the rapid expansion of CRISPR applications in biomedical research, our LNP-based delivery system represents a promising non-viral platform with broad potential for therapeutic applications, particularly in hard-to-modify cell types. STATEMENT OF SIGNIFICANCE: CRISPR/Cas9 is a transformative gene-editing technology for functional genomics and therapeutic development; however, its widespread application is constrained by the lack of safe and efficient delivery systems, particularly for hard-to-transfect cell types. Lipid nanoparticles (LNPs) represent a promising non-viral delivery strategy, yet their transfection efficiency varies substantially across cell lines due to differences in cellular membrane properties. Here, we developed and optimized an LNP platform that enables highly efficient Cas9/sgRNA-mediated gene knockout and knock-in across multiple challenging cell types, consistently outperforming commercial transfection reagents. The findings establish this LNP system as a versatile and effective non-viral platform for CRISPR/Cas9-mediated genome editing, overcoming key limitations of existing commercial lipid-based delivery agents in hard-to-modify cell types and offering a promising strategy for future clinical translation.
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CRISPR Technology Acts as a Dual-Purpose Tool in Pig Breeding: Enhancing Both Agricultural Productivity and Biomedical Applications.
Pigs have long been integral to human society for their roles in agriculture and medicine. Consequently, there is an urgent need for genetic improvement of pigs to meet human dual needs for medicine and food. In agriculture, gene editing can improve productivity traits, such as growth rate and disease resistance, which could lower farming costs and benefit consumers through enhanced meat quality. In biomedical research, gene-edited pigs offer invaluable resources as disease models and in xenotransplantation, providing organs compatible with human physiology. Currently, with CRISPR technology, especially the CRISPR/Cas9 system emerging as a transformative force in modern genetics, pigs are not only sources of sustenance but also cornerstones of biomedical innovation. This review aims to summarize the applications of CRISPR/Cas9 technology in developing pigs that serve dual roles in agriculture and biomedical applications. Compared to ZFNs and TALENs, the CRISPR/Cas9 system offers several advantages, including higher efficiency, greater specificity, ease of design and implementation, and the capability to target multiple genes simultaneously, significantly streamlining the process of genetic modifications in complex genomes. Therefore, CRISPR technology supports the enhancement of traits beneficial for agricultural productivity and facilitates applications in medicine. Furthermore, we must acknowledge the inherent deficiencies and technical challenges of the CRISPR/Cas9 technology while also anticipating emerging technologies poised to surpass CRISPR/Cas9 as the next milestones in gene editing. We hypothesize that with the continuous advancements in gene editing technologies and successful integration of traits beneficial to both agricultural productivity and medical applications, the goal of developing dual-purpose pigs for both agricultural and medical use can ultimately be achieved.
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Simple, fast and efficient iTOP-mediated delivery of CRISPR/Cas9 RNP in difficult-to-transduce human cells including primary T cells.
The advent of the CRISPR/Cas9 system has transformed the field of human genome engineering and has created new perspectives in the development of innovative cell therapies. However, the absence of a simple, fast and efficient delivery method of CRISPR/Cas9 into primary human cells has been limiting the progress of CRISPR/Cas9-based therapies. Here, we describe an optimized protocol for iTOP-mediated delivery of CRISPR/Cas9 in various human cells, including primary T cells, induced pluripotent stem cells (hiPSCs), Jurkat, ARPE-19 and HEK293 cells. We compare iTOP to other CRISPR/Cas9 delivery methods, such as electroporation and lipofection, and evaluate the corresponding gene-editing efficiencies and post-treatment cell viabilities. We demonstrate that the gene editing achieved by iTOP-mediated delivery of CRISPR/Cas9 is 40-95 % depending on the cell type, while post-iTOP cell viability remains high in the range of 70-95 %. Collectively, we present an optimized workflow for a simple, high-throughput and effective iTOP-mediated delivery of CRISPR/Cas9 to engineer difficult-to-transduce human cells. We believe that the iTOP technology® could contribute to the development of novel CRISPR/Cas9-based cell therapies.
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Characteristics, Recombination Methods, and Applications Progresses of Split-Cas9 System.
CRISPR technology has been used to revolutionize various facets of life sciences because of its potent gene editing capabilities. In particular, CRISPR technology is anticipated to be used to cure congenital disorders, and malignant cancers brought on by gene mutation. In this article, we introduce a Split-Cas9 system, in which Cas9 protein is split into two or more parts and recombined in cells to function specific induction circumstances. Split-Cas9 system can improve the therapeutic index of CRISPR technology by splitting Cas9 proteins into small fragments, thus enhancing their compatibility with virus vectors and precise temporal and spatial control. This article examined the combination mode of Split-Cas9 system, contrasted the differences in its split sites and activity efficiency, and discussed the use and clinical transformation in vivo and in vitro.
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Precision Without Selection: A Marker-Free CRISPR/Cas9-Based Protocol for Multiplexed Genome Editing in Trypanosomatids.
The CRISPR-Cas9 system has become a valuable tool for genome editing in trypanosomatid parasites such as Trypanosoma and Leishmania species. Although these organisms have been genetically engineered for a long time using homologous recombination, CRISPR/Cas9 offers improved efficiency for genome editing. However, conventional strategies employing stable Cas9 expression require the persistent use of a specific genetic background (i.e., strains expressing Cas9), depend on selectable resistance markers, compromise genomic stability, and are not readily applicable to diverse strain backgrounds. Herein, we report an optimized marker-free CRISPR/Cas9 method based on transient ribonucleoprotein (RNP) delivery that overcomes these drawbacks. Our method eliminates the need for plasmid integration or antibiotic selection while maintaining high editing efficiency. The protocol comprises the following steps: (1) design of the guide RNA (gRNA), (2) design of the repair template (cassette), (3) assembly of the ribonucleoprotein (RNP) complex, (4) delivery by electroporation, and (5) clonal screening through PCR and sequencing. The procedure permits rapid (≤3 weeks) production of homozygous mutant lines in wild-type strains, including low-density culture strains. The reproducibility and ease of the technique render it particularly suited for multiplexed editing of polyploid genomes, multi-gene families, and several different genes at once, as well as validation of the essential nature of genes. Although designed for trypanosomatids, the workflow can be adapted to other kinetoplastids, offering a flexible platform for functional genomics.
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Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13.
Currently, biology is revolutionized by ever growing applications of the CRISPR/Cas system. As discussed in this Review, new avenues have opened up for plant research and breeding by the use of the sequence-specific DNases Cas9 and Cas12 (formerly named Cpf1) and, more recently, the RNase Cas13 (formerly named C2c2). Although double strand break-induced gene editing based on error-prone nonhomologous end joining has been applied to obtain new traits, such as powdery mildew resistance in wheat or improved pathogen resistance and increased yield in tomato, improved technologies based on CRISPR/Cas for programmed change in plant genomes via homologous recombination have recently been developed. Cas9- and Cas12- mediated DNA binding is used to develop tools for many useful applications, such as transcriptional regulation or fluorescence-based imaging of specific chromosomal loci in plant genomes. Cas13 has recently been applied to degrade mRNAs and combat viral RNA replication. By the possibility to address multiple sequences with different guide RNAs and by the simultaneous use of different Cas proteins in a single cell, we should soon be able to achieve complex changes of plant metabolism in a controlled way.
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The Bibliometric Landscape of Gene Editing Innovation and Regulation in the Worldwide.
Gene editing (GE) has become one of the mainstream bioengineering technologies over the past two decades, mainly fueled by the rapid development of the CRISPR/Cas system since 2012. To date, plenty of articles related to the progress and applications of GE have been published globally, but the objective, quantitative and comprehensive investigations of them are relatively few. Here, 13,980 research articles and reviews published since 1999 were collected by using GE-related queries in the Web of Science. We used bibliometric analysis to investigate the competitiveness and cooperation of leading countries, influential affiliations, and prolific authors. Text clustering methods were used to assess technical trends and research hotspots dynamically. The global application status and regulatory framework were also summarized. This analysis illustrates the bottleneck of the GE innovation and provides insights into the future trajectory of development and application of the technology in various fields, which will be helpful for the popularization of gene editing technology.
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