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Exploration of CRISPR/Cas9-based gene editing as therapy for osteoarthritis.
OBJECTIVES: Osteoarthritis (OA) is a painful and debilitating disease and it is associated with aberrant upregulation of multiple factors, including matrix metalloproteinase 13 (MMP13), interleukin-1β (IL-1β) and nerve growth factor (NGF). In this study, we aimed to use the CRISPR/Cas9 technology, a highly efficient gene-editing tool, to study whether the ablation of OA-associated genes has OA-modifying effects. METHODS: We performed intra-articular injection of adeno-associated virus, which expressed CRISPR/Cas9 components to target each of the genes encoding MMP13, IL-1β and NGF, in a surgically induced OA mouse model. We also tested triple ablations of NGF, MMP13 and IL-1β. RESULTS: Loss-of-function of NGF palliates pain but worsens joint damage in the surgically induced OA model. Ablation of MMP13 or IL-1β reduces the expression of cartilage-degrading enzymes and attenuates structural deterioration. Targeting both MMP13 and IL-1β significantly mitigates the adverse effects of NGF blockade on the joints. CONCLUSIONS: CRISPR-mediated ablation of NGF alleviates OA pain, and deletion of MMP13-1β or IL-1β attenuates structural damage in a post-traumatic OA model. Multiplex ablations of NGF, MMP13 and IL-1β provide benefits on both pain management and joint structure maintenance. Our results suggest that CRISPR-based gene editing is useful for the identification of promising drug targets and the development of feasible therapeutic strategies for OA treatment.
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Revising, Correcting and Transferring Genes: Germline Editing Versus Natural Reproduction.
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Repurposing endogenous Type I-D CRISPR-Cas system for genome editing in Synechococcus sp. PCC7002.
Synechococcus sp. PCC7002 has been considered as a photosynthetic chassis for the conversion of CO2 into biochemicals through genetic modification. However, conventional genetic manipulation techniques prove inadequate for comprehensive genetic modifications in this strain. Here, we present the development of a genome editing tool tailored for S. PCC7002, leveraging its endogenous type I-D CRISPR-Cas system. Utilizing this novel tool, we successfully deleted the glgA1 gene and iteratively edited the genome to obtain a double mutant of glgA1 and glgA2 genes. Additionally, large DNA fragments encompassing the entire type I-A (∼14 kb) or III-B CRISPR-Cas (∼21 kb) systems were completely knocked-out in S. PCC7002 using our tool. Furthermore, the endogenous pAQ5 plasmid, approximately 38 kb in length, was successfully cured from S. PCC7002. Our work demonstrates the feasibility of harnessing the endogenous CRISPR-Cas system for genome editing in S. PCC7002, thereby enriching the genetic toolkit for this species and providing a foundation for future enhancements in its biosynthetic efficiency.
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Comparison Between Electroporation at Different Voltage Levels and Microinjection to Generate Porcine Embryos with Multiple Xenoantigen Knock-Outs.
In the context of xenotransplantation, the production of genetically modified pigs is essential. For several years, knock-out pigs were generated through somatic cell nuclear transfer employing donor cells with the desired genetic modifications, which resulted in a lengthy and cumbersome procedure. The CRISPR/Cas9 system enables direct targeting of specific genes in zygotes directly through microinjection or electroporation. However, these techniques require improvement to minimize mosaicism and low mutation rates without compromising embryo survival. This study aimed to determine the gene editing potential of these two techniques to deliver multiplexed ribonucleotide proteins (RNPs) to generate triple-knock-out porcine embryos with a multi-transgenic background. We designed RNP complexes targeting the major porcine xenoantigens GGTA1, CMAH, and B4GALNT2. We then compared the development of mosaicism and gene editing efficiencies between electroporation and microinjection. Our results indicated a significant effect of voltage increase on molecule intake in electroporated embryos, without it notably affecting the blastocyst formation rate. Our gene editing analysis revealed differences among delivery approaches and gene loci. Notably, employing electroporation at 35 V yielded the highest frequency of biallelic disruptions. However, mosaicism was the predominant genetic variant in all RNP delivery methods, underscoring the need for further research to optimize multiplex genome editing in porcine zygotes.
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Identification of Drug Resistance Mechanisms Using Genome-Wide CRISPR-Cas9 Screens.
CRISPR-Cas9 genome editing provides a means for simple and scalable production of gene knockouts in mammalian cell lines. The development of guide RNA (gRNA) libraries targeting tens of thousands of genes has allowed researchers to produce pools of cells, each containing a single gene knockout for use in genetic screens. In addition to assessing the effect of gene knockout on cell proliferation, CRISPR-Cas9 genetic screens can be used to assess gene-drug interactions. Here, we outline a protocol for performing positive and negative selection genome-wide CRISPR-Cas9 screens for identifying gene knockouts that cause drug resistance and hypersensitivity. This protocol is designed for the use of the TKOv3 library in human cell lines, but can be readily adapted for different libraries.
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Pediatric Liver Diseases: Next-Generation Therapies.
With recent advances in molecular medicine, there has been a transformation of the landscape of pediatric liver disease therapeutics. The advent of gene editing technologies, RNA therapeutics, and molecular chaperone therapies has led to precise targeting of liver pathology. This review aims to shed light on recent breakthroughs in liver-directed nucleic acid therapies such as small interfering RNA, mRNA editing, and CRISPR-based approaches, with a special focus on their application in alpha-1 antitrypsin deficiency, hepatitis B, cystic fibrosis-related liver disease, metabolic dysfunction-associated steatotic liver disease, Alagille syndrome, progressive familial intrahepatic cholestasis, and acute hepatic porphyria.
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Nanotechnological approaches for the targeted delivery of CRISPR-Cas systems for genomic modifications, biomolecular sensing, and precision medicine.
The integration strategies of CRISPR-Cas gene editing systems with nanotechnological approaches have achieved synergistic effects in targeting genes; correcting genetic disorders; and treating, sensing, and diagnosing a variety of cancers and metabolic, immunological, and complex infectious diseases-all having connectivity with distinct genetic cues and mutations. Numerous recent studies have demonstrated the use of the nano-scale properties of nanomaterials to tremendously improve the genomic-editing efficiencies of CRISPR/Cas systems for achieving 50% enhanced bioavailability, improved cell targetability, and gene-level specificity while minimizing immunogenicity, compared with conventional/ordinary delivery techniques. Thus, nano-delivery methods utilizing the unique properties of nanomaterials, molecular interactions, biocompatibility, targeted cellular uptake, and nuclear delivery capability effectively overcame the challenges of inefficient biomolecular delivery, and off-target effects were effectively overcome. Nano -carriers made up of materials such as DNA lattices, lipids, dendrimers, polymers, peptides, and metals (gold, silver, etc.) that were explored for facilitating the precise delivery of CRISPR/Cas components, sensing biomolecules, and diagnostic purposes are discussed in this review report. The ability of DNA scaffold materials to incorporate nano-CRISPR systems, to sense biomolecules, and for targeted cellular delivery of payloads (e.g., Cas9, Cas12, Cas13, and Cas14 proteins and single-guide RNAs (sgRNAs)) maximized gene targeting and improved therapeutic outcomes while achieving up to 90% efficiency compared with common/trivial delivery methods.
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'CAMERA' records cell action with new CRISPR tricks.
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CRISPR-Cas systems in enterococci.
Enterococci are members of the microbiota of humans and other animals. They can also be found in the environment, associated with food, healthcare infections, and hospital settings. Due to their wide distribution, they are inserted in the One Health context. The selective pressure caused by the extensive use of antimicrobial agents in humans, animals, and agriculture has increased the frequency of resistance to various drugs among enterococcal species. CRISPR-Cas system, an important prokaryotic defense mechanism against the entry of mobile genetic elements, may prevent the acquisition of genes involved in antimicrobial resistance and virulence. This system has been increasingly used as a gene editing tool, which can be used as a way to recognize and inactivate genes of interest. Here, we conduct a review on CRISPR systems found in enterococci, considering their occurrence, structure and organization, mechanisms of action and use as a genetic engineering technology. Type II-A CRISPR-Cas systems were shown to be the most frequent among enterococcal species, and the orphan CRISPR2 was the most commonly found system (54.1%) among enterococcal species, especially in Enterococcus faecalis. Distribution of CRISPR systems varied among species. CRISPR systems had 1 to 20 spacers, with size between 23 and 37 bp and direct repeat sequences from 25 to 37 bp. Several applications of the CRISPR-Cas biotechnology have been described in enterococci, mostly in vitro, using this editing tool to target resistance- and virulence-related genes.
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Efficient and stable CRISPR/Cas9-mediated genome-editing of human type 2 innate lymphoid cells.
Innate lymphoid cells (ILCs) are a family of innate lymphocytes with important roles in immune response coordination and maintenance of tissue homeostasis. The ILC family includes group 1 (ILC1s), group 2 (ILC2s) and group 3 (ILC3s) 'helper' ILCs, as well as cytotoxic Natural Killer (NK) cells. Study of helper ILCs in humans presents several challenges, including their low proportions in peripheral blood or needing access to rare samples to study tissue resident ILC populations. In addition, the lack of established protocols harnessing genetic manipulation platforms has limited the ability to explore molecular mechanism regulating human helper ILC biology. CRISPR/Cas9 is an efficient genome editing tool that enables the knockout of genes of interest, and is commonly used to study molecular regulation of many immune cell types. Here, we developed methods to efficiently knockout genes of interest in human ILC2s. We discuss challenges and lessons learned from our CRISPR/Cas9 gene editing optimizations using a nucleofection transfection approach and test a range of conditions and nucleofection settings to obtain a protocol that achieves effective and stable gene knockout while maintaining optimal cell viability. Using IL-4 as a representative target, we compare different ribonucleoprotein configurations, as well as assess effects of length of time in culture and other parameters that impact CRISPR/Cas9 transfection efficiency. Collectively, we detail a CRISPR/Cas9 protocol for efficient genetic knockout to aid in studying molecular mechanism regulating human ILC2s.
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