CRISPR-Based Genome Editing in Translational Research—Second Edition

A special issue of Cells (ISSN 2073-4409). This special issue belongs to the section "Cell and Gene Therapy".

Deadline for manuscript submissions: 31 August 2024 | Viewed by 5584

Special Issue Editors

Dr. Jifeng Zhang
E-Mail Website
Guest Editor
Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical School, Ann Arbor, MI 48109, USA
Interests: https://camtrast.med.umich.edu/about/people/jifeng-zhang-phd
Special Issues, Collections and Topics in MDPI journals
Center for Advanced Models and Translational Sciences and Therapeutics, University of Michigan Medical Center, Ann Arbor, MI 48109, USA
Interests: genome; gene editing; cell biology; CRISPR/Cas9
Special Issues, Collections and Topics in MDPI journals
Center for Advanced Models and Translational Sciences and Therapeutics, University of Michigan Medical Center, Ann Arbor, MI 48109, USA
Interests: genome; gene editing; cell biology; CRISPR/Cas9
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Emerging gene editing tools represented by CRISPR/Cas9 have had a significant impact on the translational biomedical research field. Thanks to their ease of use and high efficiency, they are widely used for the production of novel animal and cellular models and for the development of gene-editing-based therapy for genetic and nongenetic diseases. At the same time, many roadblocks remain in the path toward their eventual clinical applications, such as substantial off-target editing events which need to be minimized and a lack of efficient and safe in vivo delivery methods.

This Special Issue calls for research and review articles on any topics related to the translational application of CRISPR in modern biomedical research.

Dr. Jifeng Zhang
Dr. Dongshan Yang
Dr. Jie Xu
Guest Editors

Manuscript Submission Information

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Published Papers (4 papers)

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Research

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14 pages, 1842 KiB  
Article
Simplifying Genotyping of Mutants from Genome Editing with a Parallel qPCR-Based iGenotype Index
Cells 2024, 13(3), 247; https://doi.org/10.3390/cells13030247 - 29 Jan 2024
Viewed by 515
Abstract
Targeted genome editing is a powerful tool in reverse genetic studies of gene function in many aspects of biological and pathological processes. The CRISPR/Cas system or engineered endonucleases such as ZFNs and TALENs are the most widely used genome editing tools that are [...] Read more.
Targeted genome editing is a powerful tool in reverse genetic studies of gene function in many aspects of biological and pathological processes. The CRISPR/Cas system or engineered endonucleases such as ZFNs and TALENs are the most widely used genome editing tools that are introduced into cells or fertilized eggs to generate double-strand DNA breaks within the targeted region, triggering cellular DNA repair through either homologous recombination or non-homologous end joining (NHEJ). DNA repair through the NHEJ mechanism is usually error-prone, leading to point mutations or indels (insertions and deletions) within the targeted region. Some of the mutations in embryos are germline transmissible, thus providing an effective way to generate model organisms with targeted gene mutations. However, point mutations and short indels are difficult to be effectively genotyped, often requiring time-consuming and costly DNA sequencing to obtain reliable results. Here, we developed a parallel qPCR assay in combination with an iGenotype index to allow simple and reliable genotyping. The genotype-associated iGenotype indexes converged to three simple genotype-specific constant values (1, 0, −1) regardless of allele-specific primers used in the parallel qPCR assays or gene mutations at wide ranges of PCR template concentrations, thus resulting in clear genotype-specific cutoffs, established through statistical analysis, for genotype identification. While we established such a genotyping assay in the Xenopus tropicalis model, the approach should be applicable to genotyping of any organism or cells and can be potentially used for large-scale, automated genotyping. Full article
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15 pages, 4193 KiB  
Article
Generation and Characterization of a Zebrafish Model for ADGRV1-Associated Retinal Dysfunction Using CRISPR/Cas9 Genome Editing Technology
Cells 2023, 12(12), 1598; https://doi.org/10.3390/cells12121598 - 10 Jun 2023
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Abstract
Worldwide, around 40,000 people progressively lose their eyesight as a consequence of retinitis pigmentosa (RP) caused by pathogenic variants in the ADGRV1 gene, for which currently no treatment options exist. A model organism that mimics the human phenotype is essential to unravel the [...] Read more.
Worldwide, around 40,000 people progressively lose their eyesight as a consequence of retinitis pigmentosa (RP) caused by pathogenic variants in the ADGRV1 gene, for which currently no treatment options exist. A model organism that mimics the human phenotype is essential to unravel the exact pathophysiological mechanism underlying ADGRV1-associated RP, and to evaluate future therapeutic strategies. The introduction of CRISPR/Cas-based genome editing technologies significantly improved the possibilities of generating mutant models in a time- and cost-effective manner. Zebrafish have been recognized as a suitable model to study Usher syndrome-associated retinal dysfunction. Using CRISPR/Cas9 technology we introduced a 4bp deletion in adgrv1 exon 9 (adgrv1rmc22). Immunohistochemical analysis showed that Adgrv1 was absent from the region of the photoreceptor connecting cilium in the adgrv1rmc22 zebrafish retina. Here, the absence of Adgrv1 also resulted in reduced levels of the USH2 complex members usherin and Whrnb, suggesting that Adgrv1 interacts with usherin and Whrnb in zebrafish photoreceptors. When comparing adgrv1rmc22 zebrafish with wild-type controls, we furthermore observed increased levels of aberrantly localized rhodopsin in the photoreceptor cell body, and decreased electroretinogram (ERG) B-wave amplitudes which indicate that the absence of Adgrv1 results in impaired retinal function. Based on these findings we present the adgrv1rmc22 zebrafish as the first ADGRV1 mutant model that displays an early retinal dysfunction. Moreover, the observed phenotypic changes can be used as quantifiable outcome measures when evaluating the efficacy of future novel therapeutic strategies for ADGRV1-associated RP. Full article
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Review

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14 pages, 1200 KiB  
Review
CRISPR-Cas System Is an Effective Tool for Identifying Drug Combinations That Provide Synergistic Therapeutic Potential in Cancers
Cells 2023, 12(22), 2593; https://doi.org/10.3390/cells12222593 - 09 Nov 2023
Viewed by 845
Abstract
Despite numerous efforts, the therapeutic advancement for neuroblastoma and other cancer treatments is still ongoing due to multiple challenges, such as the increasing prevalence of cancers and therapy resistance development in tumors. To overcome such obstacles, drug combinations are one of the promising [...] Read more.
Despite numerous efforts, the therapeutic advancement for neuroblastoma and other cancer treatments is still ongoing due to multiple challenges, such as the increasing prevalence of cancers and therapy resistance development in tumors. To overcome such obstacles, drug combinations are one of the promising applications. However, identifying and implementing effective drug combinations are critical for achieving favorable treatment outcomes. Given the enormous possibilities of combinations, a rational approach is required to predict the impact of drug combinations. Thus, CRISPR-Cas-based and other approaches, such as high-throughput pharmacological and genetic screening approaches, have been used to identify possible drug combinations. In particular, the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats) is a powerful tool that enables us to efficiently identify possible drug combinations that can improve treatment outcomes by reducing the total search space. In this review, we discuss the rational approaches to identifying, examining, and predicting drug combinations and their impact. Full article
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25 pages, 2004 KiB  
Review
Clinical Approaches for Mitochondrial Diseases
Cells 2023, 12(20), 2494; https://doi.org/10.3390/cells12202494 - 20 Oct 2023
Cited by 1 | Viewed by 2180
Abstract
Mitochondria are subcontractors dedicated to energy production within cells. In human mitochondria, almost all mitochondrial proteins originate from the nucleus, except for 13 subunit proteins that make up the crucial system required to perform ‘oxidative phosphorylation (OX PHOS)’, which are expressed by the [...] Read more.
Mitochondria are subcontractors dedicated to energy production within cells. In human mitochondria, almost all mitochondrial proteins originate from the nucleus, except for 13 subunit proteins that make up the crucial system required to perform ‘oxidative phosphorylation (OX PHOS)’, which are expressed by the mitochondria’s self-contained DNA. Mitochondrial DNA (mtDNA) also encodes 2 rRNA and 22 tRNA species. Mitochondrial DNA replicates almost autonomously, independent of the nucleus, and its heredity follows a non-Mendelian pattern, exclusively passing from mother to children. Numerous studies have identified mtDNA mutation-related genetic diseases. The consequences of various types of mtDNA mutations, including insertions, deletions, and single base-pair mutations, are studied to reveal their relationship to mitochondrial diseases. Most mitochondrial diseases exhibit fatal symptoms, leading to ongoing therapeutic research with diverse approaches such as stimulating the defective OXPHOS system, mitochondrial replacement, and allotropic expression of defective enzymes. This review provides detailed information on two topics: (1) mitochondrial diseases caused by mtDNA mutations, and (2) the mechanisms of current treatments for mitochondrial diseases and clinical trials. Full article
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