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A new patient-derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of α-dystroglycan.
Dystroglycan, an extracellular matrix receptor, has essential functions in various tissues. Loss of α-dystroglycan-laminin interaction due to defective glycosylation of α-dystroglycan underlies a group of congenital muscular dystrophies often associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic human dystroglycanopathy cell models has limited our ability to test potential drugs in a human- and neural-specific context. Here, we generated induced pluripotent stem cells (iPSCs) from a severe dystroglycanopathy patient with homozygous FKRP (fukutin-related protein gene) mutation. We showed that CRISPR/Cas9-mediated gene correction of FKRP restored glycosylation of α-dystroglycan in iPSC-derived cortical neurons, whereas targeted gene mutation of FKRP in wild-type cells disrupted this glycosylation. In parallel, we screened 31,954 small molecule compounds using a mouse myoblast line for increased glycosylation of α-dystroglycan. Using human FKRP-iPSC-derived neural cells for hit validation, we demonstrated that compound 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile (4BPPNit) significantly augmented glycosylation of α-dystroglycan, in part through upregulation of LARGE1 glycosyltransferase gene expression. Together, isogenic human iPSC-derived cells represent a valuable platform for facilitating dystroglycanopathy drug discovery and therapeutic development.
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Genome editing by miniature CRISPR/Cas12f1 enzyme in Escherichia coli.
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system is a valuable genome editing tool for microorganisms. However, the commonly used Cas9 nuclease derived from Streptococcus pyogenes (SpCas9) is not applicable to many industrially relevant bacteria, due to its cytotoxicity and large size (1368 amino acids [aa]). We developed an alternative genome editing system using a miniature Cas12f1 nuclease (529 aa) derived from an uncultured archaeon, Un1Cas12f1. When editing four dispensable genes in Escherichia coli MG1655 and BW25113, the CRISPR/Un1Cas12f1 system showed higher efficiency (63%-100%) than the CRISPR/SpCas9 system (50%-79%). The CRISPR/Un1Cas12f1 genome editing system is expected to be applied to the genome editing of a wide variety of bacteria.
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[Prospecting application of CRISPR/Cas9 genome editing technology in research of medicinal plants].
CRISPR/Cas9 genome editing technology is a newly discovered genome editing technology in recent years.It has been widely used in many fields such as gene therapy, gene function research, animal model-making,cropvariety improvement and so on. This article briefly introduces the CRISPR/Cas9 genome editing system, and discusses its potential applications in the research of medicinal plants, including functional genomics studies, secondary metabolism and synthetic biology research of important active components and molecular breeding research. CRISPR/Cas9 genome editing technology provides a new method for the research of medicinal plants.
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Efficient Genome Editing Using 'NanoMEDIC' AsCas12a-VLPs Produced with Pol II-Transcribed crRNA.
Virus-like particles (VLPs) are an attractive vehicle for the delivery of Cas nuclease and guide RNA ribonucleoprotein complexes (RNPs). Most VLPs are produced by packaging SpCas9 and its sgRNA, which is expressed from the RNA polymerase III (Pol III)-transcribed U6 promoter. VLPs assemble in the cytoplasm, but U6-driven sgRNA is localized in the nucleus, which hinders the efficient formation and packaging of RNPs into VLPs. In this study, using the nuclease packaging mechanism of 'NanoMEDIC' VLPs, we produced VLPs with AsCas12a and exploited its ability to process pre-crRNA. This allowed us to direct crRNA in the cytoplasm as part of a Pol II-driven transcript where AsCas12a excised mature crRNA, thus boosting RNP incorporation into VLPs. CMV-driven crRNA increased Venus and CCR5 transgene knockout levels in 293 cells from 30% to 50-90% and raised the level of endogenous CXCR4 knockout in Jurkat T cells from 1% to 20%. Changing a single crRNA to an array of three or six identical crRNAs improved CXCR4 knockout rates by up to 60-70%. Compared to SpCas9-VLPs, the editing efficiencies of AsCas12a-VLPs were higher, regardless of promoter usage. Thus, we showed that AsCas12a and CMV-driven crRNA could be efficiently packaged into VLPs and mediate high levels of gene editing. AsCas12a-VLPs are a new and promising tool for the delivery of RNPs into mammalian cells that will allow efficient target genome editing and may be useful for gene therapy applications.
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Genome editing reagent delivery in plants.
Genome editing holds the potential for rapid crop improvement to meet the challenge of feeding the planet in a changing climate. The delivery of gene editing reagents into the plant cells has been dominated by plasmid vectors delivered using agrobacterium or particle bombardment. This approach involves the production of genetically engineered plants, which need to undergo regulatory approvals. There are various reagent delivery approaches available that have enabled the delivery of DNA-free editing reagents. They invariably involve the use of ribonucleoproteins (RNPs), especially in the case of CRISPR/Cas9-mediated gene editing. The explant of choice for most of the non-DNA approaches utilizes protoplasts as the recipient explant. While the editing efficiency is high in protoplasts, the ability to regenerate individual plants from edited protoplasts remains a challenge. There are various innovative delivery approaches being utilized to perform in planta edits that can be incorporated in the germline cells or inherited via seed. With the modification and adoption of various novel approaches currently being used in animal systems, it seems likely that non-transgenic genome editing will become routine in higher plants.
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Effective gene editing of melanoma by delivery of Cas9 mRNA with highly branched poly(β-amino ester).
Melanoma, the most aggressive form of skin cancer, still lacks clinically reliable treatments. CRISPR/Cas9 gene editing offers a promising approach for melanoma therapy; however, the efficient and safe delivery of CRISPR/Cas9 components to melanoma cells remains a formidable challenge. Here, we report the development of highly branched poly(β-amino ester) (HPAE) for the efficient delivery of Cas9 mRNA to melanoma cells and for transdermal application in vivo. HPAE is synthesized via a one-pot "A2 + B4 + C2" type Michael addition strategy and is capable of efficiently condensing mRNA to form nanosized polyplexes with a moderate positive charge. In human embryonic kidney cells (HEK293T), mouse melanoma cells (B16F10), and human melanoma cells (A2058 and A375), up to 76.3%, 74.6%, 16.5%, and 57.1% mRNA transfection efficiency was achieved without obvious cytotoxicity. Moreover, repeated dosing further enhanced mRNA transfection efficiency, resulting in sustained gene expression. Importantly, HPAE enables the effective co-delivery of Cas9 mRNA and sgRNA to melanoma cells, facilitating up to 25.5% gene knockout. HPAE also demonstrates strong transdermal capability, resulting in high in vivo mRNA expression following direct topical administration. This study demonstrates the feasibility of treating melanoma through the transdermal application of the CRISPR/Cas9 system, enabled by the efficient mRNA delivery provided by HPAE.
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Applications of CRISPR/Cas as a Toolbox for Hepatitis B Virus Detection and Therapeutics.
Hepatitis B virus (HBV) infection remains a significant global health challenge, leading to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Covalently closed circular DNA (cccDNA) and integrated HBV DNA are pivotal in maintaining viral persistence. Recent advances in CRISPR/Cas technology offer innovative strategies to inhibit HBV by directly targeting both cccDNA and integrated HBV DNA or indirectly by degrading HBV RNAs or targeting host proteins. This review provides a comprehensive overview of the latest advancements in using CRISPR/Cas to inhibit HBV, with a special highlight on newer non-double-strand (non-DSB) break approaches. Beyond the canonical use of CRISPR/Cas for target inhibition, we discuss additional applications, including HBV diagnosis and developing models to understand cccDNA biology, highlighting the diverse use of this technology in the HBV field.
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Applications of Functional Genomics for Drug Discovery.
Many diseases, such as diabetes, autoimmune diseases, cancer, and neurological disorders, are caused by a dysregulation of a complex interplay of genes. Genome-wide association studies have identified thousands of disease-linked polymorphisms in the human population. However, detailing the causative gene expression or functional changes underlying those associations has been elusive in many cases. Functional genomics is an emerging field of research that aims to deconvolute the link between genotype and phenotype by making use of large -omic data sets and next-generation gene and epigenome editing tools to perturb genes of interest. Here we review how functional genomic tools can be used to better understand the biological interplay between genes, improve disease modeling, and identify novel drug targets. Incorporation of functional genomic capabilities into conventional drug development pipelines is predicted to expedite the development of first-in-class therapeutics.
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Genome editing: intellectual property and product development in plant biotechnology.
Genome editing is a revolutionary technology in molecular biology. While scientists are fascinated with the unlimited possibilities provided by directed and controlled changes in DNA in eukaryotes and have eagerly adopted such tools for their own experiments, an understanding of the intellectual property (IP) implications involved in bringing genome editing-derived products to market is often lacking. Due to the ingenuity of genome editing, the time between new product conception and its actual existence can be relatively short; therefore knowledge about IP of the various genome editing methods is relevant. This point must be regarded in a national framework as patents are instituted nationally. Therefore, when designing scientific work that could lead to a product, it is worthwhile to consider the different methods used for genome editing not only for their scientific merits but also for their compatibility with a speedy and reliable launch into the desired market.
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Off-target Cas9 crystallized.
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