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Challenges to Gene Editing Approaches in the Retina.
Retinal gene therapy has recently been at the cutting edge of clinical development in the diverse field of genetic therapies. The retina is an attractive target for genetic therapies such as gene editing due to the distinctive anatomical and immunological features of the eye, known as immune privilege, so that inherited retinal diseases (IRDs) have been studied in several clinical studies. Thus, rapid strides are being made toward developing targeted treatments for IRDs. Gene editing in the retina faces a group of heterogenous challenges, including editing efficiencies, off-target effects, the anatomy of the target organ, immune responses, inactivation, and identifying optimal application methods. As clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) based technologies are at the forefront of current gene editing advances, their specific editing efficiency challenges and potential off-target effects were assessed. The immune privilege of the eye reduces the likelihood of systemic immune responses following retinal gene therapy, but possible immune responses must not be discounted. Immune responses to gene editing in the retina may be humoral or cell mediated, with immunologically active cells, including microglia, implicated in facilitating possible immune responses to gene editing. Immunogenicity of gene therapeutics may also lead to the inactivation of edited cells, reducing potential therapeutic benefits. This review outlines the broad spectrum of potential challenges currently facing retinal gene editing, with the goal of facilitating further advances in the safety and efficacy of gene editing therapies.
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CRISPR/Cas genome editing, functional genomics, and diagnostics for parasitic helminths.
Functional genomics using CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated endonuclease)-based approaches has revolutionized biomedical sciences. Gene editing is also widespread in parasitology generally and its use is increasing in studies on helminths including flatworm and roundworm parasites. Here, we survey the progress, specifically with experimental CRISPR-facilitated functional genomics to investigate helminth biology and pathogenesis, and also with the burgeoning use of CRISPR-based methods to assist in diagnosis of helminth infections. We also provide an historical timeline of the introduction and uses of CRISPR in helminth species to date.
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The CRISPR-Cas revolution in head and neck cancer: a new era of targeted therapy.
Head and neck cancer (HNC) encompasses a diverse array of malignancies impacting the anatomical structures of the head and neck region, ranking as the seventh most prevalent cancer type globally. The occurrence and advancement of HNC are intricately linked to mutations and disruptions within critical signaling pathways, accentuating the imperative for targeted therapeutic interventions to rectify these genetic aberrations. Traditional treatment modalities, including surgical intervention and adjuvant chemotherapy or radiotherapy, frequently culminate in considerable morbidity and suboptimal prognoses. Recently, the CRISPR-Cas system has emerged as a revolutionary gene-editing platform, poised to redefine therapeutic approaches in gene therapy and oncological research. Despite its potential, CRISPR-Cas faces challenges such as off-target effects, delivery inefficiencies, and immunogenicity, which must be addressed for clinical success. This review meticulously evaluates the progress in CRISPR-Cas technologies aimed at targeting essential signaling pathways implicated in HNC, addressing current challenges while highlighting optimal targets, Cas nucleases, and innovative delivery mechanisms, thereby elucidating the therapeutic potential and clinical applicability of the CRISPR-Cas paradigm in the management of HNC.
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Production of Purified CasRNPs for Efficacious Genome Editing.
CRISPR-Cas systems have been harnessed as modular genome editing reagents for functional genomics and show promise to cure genetic diseases. Directed by a guide RNA, a Cas effector introduces a double stranded break in DNA and host cell DNA repair leads to the introduction of errors (e.g., to knockout a gene) or a programmed change. Introduction of a Cas effector and guide RNA as a purified Cas ribonucleoprotein complex (CasRNP) has recently emerged as a powerful approach to alter cell types and organisms. Not only does CasRNP editing exhibit increased efficacy and specificity, it avoids optimization and iteration of species-specific factors such as codon usage, promoters, and terminators. CasRNP editing has been rapidly adopted for research use in many contexts and is quickly becoming a popular method to edit primary cells for therapeutic application. This article describes how to make a Cas9 RNP and outlines its use for gene editing in human cells. © 2017 by John Wiley & Sons, Inc.
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Nuclease-mediated genome editing: At the front-line of functional genomics technology.
Genome editing with engineered endonucleases is rapidly becoming a staple method in developmental biology studies. Engineered nucleases permit random or designed genomic modification at precise loci through the stimulation of endogenous double-strand break repair. Homology-directed repair following targeted DNA damage is mediated by co-introduction of a custom repair template, allowing the derivation of knock-out and knock-in alleles in animal models previously refractory to classic gene targeting procedures. Currently there are three main types of customizable site-specific nucleases delineated by the source mechanism of DNA binding that guides nuclease activity to a genomic target: zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR). Among these genome engineering tools, characteristics such as the ease of design and construction, mechanism of inducing DNA damage, and DNA sequence specificity all differ, making their application complementary. By understanding the advantages and disadvantages of each method, one may make the best choice for their particular purpose.
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[CRISPR-Cas9 for muscle dystrophies].
Muscular dystrophies are a group of rare muscular disorders characterized by weakness and progressive degeneration of the muscle. They are diseases of genetic origin caused by the mutation of one or more genes involved in muscle function. Despite significant progress made in the field of biotherapies in recent years, there is as yet no curative treatment available for these diseases. Studies conducted since the discovery of the CRISPR-Cas9 genomic editing tool have nevertheless led to significant and promising advances in the treatment of muscular dystrophies. CRISPR-Cas9 system allows a stable and permanent edition of the genome and should make it possible to avoid long, partially efficient and repetitive treatments. In this review, we will discuss the latest therapeutic advances obtained using the CRISPR-Cas9 system in genetic muscular dystrophies.
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Single-strand template repair: key insights to increase the efficiency of gene editing.
DNA double-strand breaks (DSBs) pose a serious hazard for the stability of the genome. CRISPR-Cas9-mediated gene editing intentionally creates a site-specific DSB to modify the genomic sequence, typically from an introduced single-stranded DNA donor. However, unlike typical forms of homologous recombination, single-strand template repair (SSTR) is Rad51-independent. Moreover, this pathway is distinct from other previously characterized Rad51-independent processes. Here, we briefly review the work characterizing this pathway, and how these findings can be used to guide and improve current gene editing strategies.
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Brain-Targeted Cas12a Ribonucleoprotein Nanocapsules Enable Synergetic Gene Co-Editing Leading to Potent Inhibition of Orthotopic Glioblastoma.
Gene-editing technology shows great potential in glioblastoma (GBM) therapy. Due to the complexity of GBM pathogenesis, a single gene-editing-based therapy is unlikely to be successful; therefore, a multi-gene knockout strategy is preferred for effective GBM inhibition. Here, a non-invasive, biodegradable brain-targeted CRISPR/Cas12a nanocapsule is used that simultaneously targeted dual oncogenes, EGFR and PLK1, for effective GBM therapy. This cargo nanoencapsulation technology enables the CRISPR/Cas12a system to achieve extended blood half-life, efficient blood-brain barrier (BBB) penetration, active tumor targeting, and selective release. In U87MG cells, the combinatorial gene editing system resulted in 61% and 33% knockout of EGFR and PLK1, respectively. Following systemic administration, the CRISPR/Cas12a system demonstrated promising brain tumor accumulation that led to extensive EGFR and PLK1 gene editing in both U87MG and patient-derived GSC xenograft mouse models with negligible off-target gene editing detected through NGS. Additionally, CRISPR/Cas12a nanocapsules that concurrently targeted the EGFR and PLK1 oncogenes showed superior tumor growth suppression and significantly improved the median survival time relative to nanocapsules containing single oncogene knockouts, signifying the potency of the multi-oncogene targeting strategy. The findings indicate that utilization of the CRISPR/Cas12a combinatorial gene editing technique presents a practical option for gene therapy in GBM.
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Efficient Targeted Mutagenesis in Brassica Crops Using CRISPR/Cas Systems.
CRISPR/Cas has been established for targeted mutagenesis in many plant species since 2013, including Brassica napus and Brassica oleracea. Since that time, improvements have been made in terms of efficiency and choice of CRISPR systems. This protocol encompasses improved Cas9 efficiency and an alternative Cas12a system, allowing more challenging and diverse editing outcomes to be achieved.
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Harnessing the Streptomyces-originating type I-E CRISPR/Cas system for efficient genome editing in Streptomyces.
Since their discovery, CRISPR/Cas systems have significantly expanded the genetic toolbox, aiding in the exploration and enhanced production of natural products across various microbes. Among these, class 2 CRISPR/Cas systems are simpler and more broadly used, but they frequently fail to function effectively in many Streptomyces strains. In this study, we present an engineered class 1 type I CRISPR/Cas system derived from Streptomyces avermitilis, which enables efficient gene editing in phylogenetically distant Streptomyces strains. Through a plasmid interference assay, we identified the effective protospacer adjacent motif as 5'-AAN-3'. Utilizing this system, we achieved targeted chromosomal deletions ranging from 8 bp to 100 kb, with efficiencies exceeding 92%. We further utilized this system to insert DNA fragments into different Streptomyces genomes, facilitating the heterologous expression of exogenous genes and the activation of endogenous natural product biosynthetic gene clusters. Overall, we established a type I CRISPR/Cas-based gene-editing methodology that significantly advances the exploration of Streptomyces, known for their rich natural product resources. This is the first report of a gene editing tool developed based on the endogenous class 1 type I CRISPR/Cas system in Streptomyces spp. Our work enriches the Streptomyces gene manipulation toolbox and advances the discovery of valuable natural products within these organisms.
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