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Gene Therapy in Tyrosinemia: Potential and Pitfalls.
In this chapter, we intend to review gene therapy concepts applied to the potential treatment of tyrosinemia for parents and pediatricians. Therefore, our main objective is to give general informations in a comprehensible manner. Considering the nature of tyrosinemia and the current state of technology, a particular focus will be put on strategies using viral delivery of DNA to the liver. In light of the recent development of the CRISPR technology and the revival of promises for previously unavailable therapeutical tools, the present chapter aims at presenting up to date facts and potential pitfalls towards an application for metabolic diseases, in particular tyrosinemia.
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Genotyping MUltiplexed-Sequencing of CRISPR-Localized Editing (GMUSCLE): An Experimental and Computational Approach for Analyzing CRISPR-Edited Cells.
Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) creates double-stranded breaks, the repair of which generates indels around the target sites. These repairs can be mono-/multi-allelic, and the editing is often random and sometimes prolonged, resulting in considerable intercellular heterogeneity. The genotyping of CRISPR-Cas9-edited cells is challenging and the traditional genotyping methods are laborious. We introduce here a streamlined experimental and computational protocol for genotyping CRISPR-Cas9 genome-edited cells including cost-effective multiplexed sequencing and the software Genotyping MUltiplexed-Sequencing of CRISPR-Localized Editing (GMUSCLE). In this approach, CRISPR-Cas9-edited products are sequenced in great depth, then GMUSCLE quantitatively and qualitatively identifies the genotypes, which enable the selection and investigation of cell clones with genotypes of interest. We validate the protocol and software by performing CRISPR-Cas9-mediated disruption on interferon-α/β receptor alpha, multiplexed sequencing, and identifying the genotypes simultaneously for 20 cell clones. Besides the multiplexed sequencing ability of this protocol, GMUSCLE is also applicable for the sequencing data from bulk cell populations. GMUSCLE is publicly available at our HGIDSOFT server and GitHub.
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The Red Flour Beetle as Model for Comparative Neural Development: Genome Editing to Mark Neural Cells in Tribolium Brain Development.
With CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) scientists working with Tribolium castaneum can now generate transgenic lines with site-specific insertions at their region of interest. We present two methods to generate in vivo imaging lines suitable for marking subsets of neurons with fluorescent proteins. The first method relies on homologous recombination and uses a 2A peptide to create a bicistronic mRNA. In such lines, the target and the marker proteins are not fused but produced at equal amounts. This work-intensive method is compared with creating gene-specific enhancer traps that do not rely on homologous recombination. These are faster to generate but reflect the expression of the target gene less precisely. Which method to choose, strongly depends on the aims of each research project and in turn impacts of how neural cells and their development are marked. We describe the necessary steps from designing constructs and guide RNAs to embryonic injection and making homozygous stocks.
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CRISPR/Cas9 gene editing in a chicken model: current approaches and applications.
Improvements in genome editing technology in birds using primordial germ cells (PGCs) have made the development of innovative era genome-edited avian models possible, including specific chicken bioreactors, production of knock-in/out chickens, low-allergenicity eggs, and disease-resistance models. New strategies, including CRISPR/Cas9, have made gene editing easy and highly efficient in comparison to the well-known process of homologous recombination. The clustered regularly interspaced short palindromic repeats (CRISPR) technique enables us to understand the function of genes and/or to modify the animal phenotype to fit a specific scientific or production target. To facilitate chicken genome engineering applications, we present a concise description of the method and current application of the CRISPR/Cas9 system in chickens. Different strategies for delivering sgRNAs and the Cas9 protein, we also present extensively. Furthermore, we describe a new gesicle technology as a way to deliver Cas9/sgRNA complexes into target cells, and we discuss the advantages and describe basal applications of the CRISPR/Cas9 system in a chicken model.
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Gene tagging via CRISPR-mediated homology-directed repair in cassava.
Research on a few model plant-pathogen systems has benefitted from years of tool and resource development. This is not the case for the vast majority of economically and nutritionally important plants, creating a crop improvement bottleneck. Cassava bacterial blight (CBB), caused by Xanthomonas axonopodis pv. manihotis (Xam), is an important disease in all regions where cassava (Manihot esculenta Crantz) is grown. Here, we describe the development of cassava that can be used to visualize one of the initial steps of CBB infection in vivo. Using CRISPR-mediated homology-directed repair (HDR), we generated plants containing scarless insertion of GFP at the 3' end of CBB susceptibility (S) gene MeSWEET10a. Activation of MeSWEET10a-GFP by the transcription activator-like (TAL) effector TAL20 was subsequently visualized at transcriptional and translational levels. To our knowledge, this is the first such demonstration of HDR via gene editing in cassava.
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Nanotechnology-Based Delivery of CRISPR/Cas9 for Cancer Treatment: A Comprehensive Review.
CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9)-mediated genome editing has emerged as a transformative tool in medicine, offering significant potential for cancer therapy because of its capacity to precisely target and alter the genetic modifications associated with the disease. However, a major challenge for its clinical translation is the safe and efficient in vivo delivery of CRISPR/Cas9 components to target cells. Nanotechnology is a promising solution to this problem. Nanocarriers, owing to their tunable physicochemical properties, can encapsulate and protect CRISPR/Cas9 components, enabling targeted delivery and enhanced cellular uptake. This review provides a comprehensive examination of the synergistic potential of CRISPR/Cas9 and nanotechnology in cancer therapy and explores their integrated therapeutic applications in gene editing and immunotherapy. A critical aspect of in vivo CRISPR/Cas9 application is to achieve effective localization at the tumor site while minimizing off-target effects. Nanocarriers can be engineered to overcome biological barriers, thereby augmenting tumor-specific delivery and facilitating intracellular uptake. Furthermore, their design allows for controlled release of the therapeutic payload, ensuring sustained efficacy and reduced systemic toxicity. The optimization of nanocarrier attributes, including size, shape, surface charge, and composition, is crucial for improving the cellular internalization, endosomal escape, and nuclear localization of CRISPR/Cas9. Moreover, surface functionalization with targeting ligands can enhance the specificity of cancer cells, leading to improved gene-editing accuracy. This review thoroughly discusses the challenges associated with in vivo CRISPR/Cas9 delivery and the innovative nanotechnological strategies employed to overcome them, highlighting their combined potential for advancing cancer treatment for clinical application.
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[Progress of researches on toxoplasmosis vaccines based on the CRISPR/Cas9 technology].
Toxoplasma gondii is an obligatory intracellular parasite which infects a variety of warm-blooded animals and causes toxoplasmosis. Toxoplasmosis seriously endangers human health and animal husbandry production. As one of the effective gene editing tools, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been widely used for knockout of genes in T. gondii. This review summarizes the applications of the CRISPR/Cas9 technology in vaccines against single- and double-gene deletion strains of T. gondii, so as to provide insights into development of toxoplasmosis vaccines.
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CRISPR-Cas based genome editing for eradication of human viruses.
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system possess a broad range of applications for genetic modification, diagnosis and treatment of infectious as well as non-infectious disease. The CRISPR-Cas system is found in bacteria and archaea that possess the Cas protein and guide RNA (gRNA). Cas9 and gRNA forms a complex to target and cleave the desired gene, providing defense against viral infections. Human immunodeficiency virus (HIV), hepatitis B virus (HBV), herpesviruses, human papillomavirus (HPV), and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) cause major life threatening diseases which cannot cure completely by drugs. This chapter describes the present strategy of CRISPR-Cas systems for altering the genomes of viruses, mostly human ones, in order to control infections.
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A Transgenic Core Facility's Experience in Genome Editing Revolution.
The use of animal models, particularly rodents, has been immensely important to nearly all aspects of biomedical research, from basic science exploration to translational discoveries into clinical applications. The transgenic core facility that provides animal model production, preservation, and recovery services has been fundamental to the success of research efforts using animals. Recent advances in genome editing technologies, especially the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) enzyme system, have transformed the tedious animal model production into a simple and effective procedure. We, as a transgenic core facility established in 1993, adopted the CRISPR/Cas9 technology in early 2014 and have experienced the dramatic shift in the practice of animal model production, from the conventional embryonic stem cell approach to the direct genomic editing in rodent embryos. In this chapter, we describe the lessons that we learned from more than 200 genome editing projects performed in this core facility within the past 3 years. We also provide the practical guidelines for efficient generation of animal models using this technology and the insights into where new technologies lead us.
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Recent Advances in Genome Editing and Bioinformatics: Addressing Challenges in Genome Editing Implementation and Genome Sequencing.
Genome-editing technology has advanced significantly since the 2020 Nobel Prize in Chemistry was awarded for the development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). While CRISPR-Cas9 has become widely used in academic research, its social implementation has lagged due to unresolved patent disputes and slower progress in gene function analysis. To address this, new approaches bypassing direct gene function analysis are needed, with bioinformatics and next-generation sequencing (NGS) playing crucial roles. NGS is essential for sequencing the genome of target species, but challenges such as data quality, genome heterogeneity, ploidy, and small individual sizes persist. Despite these issues, advancements in sequencing technologies, like PacBio high-fidelity (HiFi) long reads and high-throughput chromosome conformation capture (Hi-C), have improved genome sequencing. Bioinformatics contributes to genome editing through off-target prediction and target gene selection, both of which require accurate genome sequence information. In this review, I will give updates on the development of genome editing and bioinformatics technologies with a focus on the rapid progress in genome sequencing.
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