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Shining light on CRISPR/Cas9 therapeutics for inherited retinal diseases.
Inherited retinal diseases (IRDs), such as retinitis pigmentosa, are a heterogenous group of genetic eye diseases characterized by degeneration of photoreceptors. They are the leading cause of blindness in the working age population in high-income countries and are an ideal target for the expanding gene editing tool kit, including rapidly evolving CRISPR/Cas9 technology. In this review, we provide a comprehensive analysis of CRISPR/Cas9 technologies currently being explored as therapeutic interventions for IRDs. Given the challenges posed by the growing complexity and size of gene editing systems, the delivery of these therapeutics to the retina has necessitated innovative approaches. We review current delivery methods, including nanoparticles, virus-like particles and traditional viral vectors, highlighting their advantages and limitations. This review underscores the potential transformative impact of gene editing on genetic disease management, emphasising that advancements in these technologies, coupled with improved pre-clinical models, bring clinically safe and effective treatments for IRDs within view.
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Single-nucleotide-level mapping of DNA regulatory elements that control fetal hemoglobin expression.
Pinpointing functional noncoding DNA sequences and defining their contributions to health-related traits is a major challenge for modern genetics. We developed a high-throughput framework to map noncoding DNA functions with single-nucleotide resolution in four loci that control erythroid fetal hemoglobin (HbF) expression, a genetically determined trait that modifies sickle cell disease (SCD) phenotypes. Specifically, we used the adenine base editor ABEmax to introduce 10,156 separate A•T to G•C conversions in 307 predicted regulatory elements and quantified the effects on erythroid HbF expression. We identified numerous regulatory elements, defined their epigenomic structures and linked them to low-frequency variants associated with HbF expression in an SCD cohort. Targeting a newly discovered γ-globin gene repressor element in SCD donor CD34+ hematopoietic progenitors raised HbF levels in the erythroid progeny, inhibiting hypoxia-induced sickling. Our findings reveal previously unappreciated genetic complexities of HbF regulation and provide potentially therapeutic insights into SCD.
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CRISPR infusion eases symptoms in genetic disease.
Some patients no longer need drugs to prevent swelling.
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Lipopeptide-Based Nanosome-Mediated Delivery of Hyperaccurate CRISPR/Cas9 Ribonucleoprotein for Gene Editing.
A transient cytosolic delivery system for accurate Cas9 ribonucleoprotein is a key factor for target specificity of the CRIPSR/Cas9 toolkit. Owing to the large size of the Cas9 protein and a long negative strand RNA, the development of the delivery system is still a major challenge. Here, a size-controlled lipopeptide-based nanosome system is reported, derived from the blood-brain barrier-permeable dNP2 peptide which is capable of delivering a hyperaccurate Cas9 ribonucleoprotein complex (HypaRNP) into human cells for gene editing. Each nanosome is capable of encapsulating and delivering ≈2 HypaRNP molecules into the cytoplasm, followed by nuclear localization at 4 h post-treatment without significant cytotoxicity. The HypaRNP thus efficiently enacts endogenous eGFP silencing and editing in human embryonic kidney cells (up to 27.6%) and glioblastoma (up to 19.7% frequency of modification). The lipopeptide-based nanosome system shows superior delivery efficiency, high controllability, and simplicity, thus providing biocompatibility and versatile platform approach for CRISPR-mediated transient gene editing applications.
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Efficient genome editing in a Mozambique tilapia cell line using CAS ribonucleoprotein complexes.
Genome editing using the CRISPR/Cas system makes it possible to rapidly characterise gene function in vitro and in vivo, and provides a powerful platform through which the genetics of farmed fish can be altered to improve traits such as resistance to important pathogens. Tilapia is one of the most important farmed fish globally; however, its farming is heavily impacted by Tilapia lake virus (TiLV). The Mozambique tilapia (Oreochromis mossambicus) brain (OmB) cell line is susceptible to TiLV, making it an ideal in vitro model for studying host-pathogen interactions and mechanisms of disease resistance. To establish OmB cells as a model for gene editing in Tilapia, it is essential to optimise genome editing protocols. In this study, we optimized a CRISPR/Cas9-based genome editing system for OmB cells using ribonucleoprotein complexes. With the optimized protocol, we successfully edited two endogenous genes with efficiencies ranging from 67% to 70%. In conclusion, we establish a highly efficient CRISPR/Cas9 RNP based gene editing workflow optimized for OmB tilapia cell lines. This optimized platform will facilitate future functional genomic studies in tilapia and support the development of TiLV-resistant tilapia.
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Advances and perspectives in the application of CRISPR/Cas9 in insects.
Insects compose more than half of all living organisms on earth, playing essential roles in global ecosystems and forming complex relationships with humans. Insect research has significant biological and practical importance. However, the application of genetic manipulation technology has long been restricted to several model insects only, such as gene knockout in Drosophila, which has severely restrained the development of insect biology research. Recently, with the increase in the release of insect genome data and the introduction of the CRISPR/Cas9 system for efficient genetic modification, it has been possible to conduct meaningful functional studies in a broad array of insect species. Here, we summarize the advances in CRISPR/Cas9 in different insect species, discuss methods for its promotion, and consider its application in future insect studies. This review provides detailed information about the application of the CRISPR/Cas9 system in insect research and presents possible ways to improve its use in functional studies and insect pest control.
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Recent progress in CRISPR-Cas-system for neurological disorders.
Different neurological diseases including, Parkinson's, Alzheimer's, and Huntington's diseases extant momentous global disease burdens, affecting millions of lives for imposing a heavy disease burden on the healthcare systems. Despite various treatment strategies aimed at alleviating symptoms, treatments remain elusive and ineffective due to the disease's complexity. However, recent advancements in gene therapy via the CRISPR-Cas system offer ground-breaking and targeted treatment options. Based on a bacterial immune mechanism, the CRISPR-Cas system enables precise genome editing, allowing for the alteration of different genetic mutations and the possible cure of genetic diseases. In the context of neurological disorders, the CRISPR-Cas system shows a promising avenue by allowing researchers to conduct genome-editing which is implicated in neurodegenerative disease therapeutics. This book chapter provides an updated overview of the application of the CRISPR-Cas system for addressing target-specific therapeutic approaches for neurodegenerative disorders. Furthermore, we discuss the principles of the CRISPR-Cas mechanism, its role in modeling neurological disorders, identifying molecular targets, and developing gene-based therapies. Additionally, the chapter explores the recent clinical trials and CRISPR-Cas-mediated treatments for neurological conditions. By leveraging the accuracy and versatility of the CRISPR-Cas system, scientists can more effectively handle the genetic underpinnings of neurodegenerative diseases. Furthermore, the chapter extends the critical viewpoints on ethical considerations and technical limitations related to the clinical deployment of this revolutionizing technique.
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Identification of Drug Resistance Genes Using a Pooled Lentiviral CRISPR/Cas9 Screening Approach.
In addition to advancing the development of gene-editing therapeutics, CRISPR/Cas9 is transforming how functional genetic studies are carried out in the lab. By increasing the ease with which genetic information can be inserted, deleted, or edited in cell and organism models, it facilitates genotype-phenotype analysis. Moreover, CRISPR/Cas9 has revolutionized the speed at which new genes underlying a particular phenotype can be identified through its application in genomic screens. Arrayed high-throughput and pooled lentiviral-based CRISPR/Cas9 screens have now been used in a wide variety of contexts, including the identification of essential genes, genes involved in cancer metastasis and tumor growth, and even genes involved in viral response. This technology has also been successfully used to identify drug targets and drug resistance mechanisms. Here, we provide a detailed protocol for performing a genome-wide pooled lentiviral CRISPR/Cas9 knockout screen to identify genetic modulators of a small-molecule drug. While we exemplify how to identify genes involved in resistance to a cytotoxic histone deacetylase inhibitor, Trichostatin A (TSA), the workflow we present can easily be adapted to different types of selections and other types of exogenous ligands or drugs.
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Applications of multiplexed CRISPR-Cas for genome engineering.
The CRISPR-Cas system has become a worldwide genome editing tool for various organisms. Its precision and efficiency have facilitated basic research, drug discovery and therapeutic interventions. In contrast to other genome editing agents, CRISPR-Cas is modulated by a short guide RNA. Due to its simplicity, CRISPR-Cas is recognized as the best candidate for multiplexed genome editing. With simultaneous targeting, efficient knockout of genes with large deletions is possible. In addition, CRISPR-Cas can induce complex structural variations, such as inversions, translocations and duplications. Moreover, by utilizing engineered CRISPR-Cas proteins specialized for direct repression or activation of gene expression, one can perform multiplexed epigenetic editing. Lastly, multiplexed targeting enables killing of specific types of cells by accumulating stress mediated by simultaneous DNA damages. Here we discuss how CRISPR-based editing technologies for multiple targets are applied in recent studies.
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CRISPR-Cas9 based genome editing for defective gene correction in humans and other mammals.
Clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR/Cas9), derived from bacterial and archean immune systems, has received much attention from the scientific community as a powerful, targeted gene editing tool. The CRISPR/Cas9 system enables a simple, relatively effortless and highly specific gene targeting strategy through temporary or permanent genome regulation or editing. This endonuclease has enabled gene correction by taking advantage of the endogenous homology directed repair (HDR) pathway to successfully target and correct disease-causing gene mutations. Numerous studies using CRISPR support the promise of efficient and simple genome manipulation, and the technique has been validated in in vivo and in vitro experiments, indicating its potential for efficient gene correction at any genomic loci. In this chapter, we detailed various strategies related to gene editing using the CRISPR/Cas9 system. We also outlined strategies to improve the efficiency of gene correction via the HDR pathway and to improve viral and non-viral mediated gene delivery methods, with an emphasis on their therapeutic potential for correcting genetic disorder in humans and other mammals.
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