Special Issue "CRISPR Genome Editing"

A special issue of Cells (ISSN 2073-4409). This special issue belongs to the section "Cell Nuclei: Function, Transport and Receptors".

Deadline for manuscript submissions: closed (15 May 2020).

Special Issue Editor

Prof. Dr. Cord Brakebusch
Website
Guest Editor
Biotech Research & Innovation Centre, The University of Copenhagen; Copenhagen, Denmark
Interests: Rho GTPases; keratinocytes; mouse disease models
Special Issues and Collections in MDPI journals

Special Issue Information

Dear Colleagues,

CRISPR genome editing has managed to change, in just a few years, most areas of biomedical research. CRISPR-mediated gene knockout has replaced siRNA-mediated knockdown to test gene functions in cell lines. CRISPR-mediated genome editing in mice greatly facilitated the generation of genetically modified mice and enabled the quick generation of mice with multiple genetic alterations. Moreover, as CRISPR genome editing is not restricted to mice, it significantly widened the spectrum of laboratory animals that can be used to study the effect of targeted mutations. Finally, CRISPR genome editing created novel ways to repair gene defects of human patients ex vivo or in vivo.

However, the shiny new world of CRISPR is not without its challenges. The biggest shortcomings right now are, first, the relative low efficiency of precise homology directed repair compared to the error-prone nonhomologous end joining repair; secondly, the presence of off-target mutations; and thirdly, the often insufficient effectiveness of introducing CRISPR nucleases together with targeting templates into stem cells. This Special Issue of Cells is therefore dedicated to reviews and original articles describing efforts to overcome the bottlenecks of this fascinating genome editing technology and to present the state-of-the-art in that field.

Prof. Cord Brakebusch
Guest Editor

Manuscript Submission Information

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Keywords

  • CRISPR
  • homology dependent repair
  • Cas9
  • genetically modified mice
  • genome editing

Published Papers (5 papers)

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Research

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Open AccessArticle
High-Capacity Adenoviral Vectors Permit Robust and Versatile Testing of DMD Gene Repair Tools and Strategies in Human Cells
Cells 2020, 9(4), 869; https://doi.org/10.3390/cells9040869 - 02 Apr 2020
Cited by 1
Abstract
Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle wasting disorder arising from mutations in the ~2.4 Mb dystrophin-encoding DMD gene. RNA-guided CRISPR-Cas9 nucleases (RGNs) are opening new DMD therapeutic routes whose bottlenecks include delivering sizable RGN complexes for assessing their effects on [...] Read more.
Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle wasting disorder arising from mutations in the ~2.4 Mb dystrophin-encoding DMD gene. RNA-guided CRISPR-Cas9 nucleases (RGNs) are opening new DMD therapeutic routes whose bottlenecks include delivering sizable RGN complexes for assessing their effects on human genomes and testing ex vivo and in vivo DMD-correcting strategies. Here, high-capacity adenoviral vectors (HC-AdVs) encoding single or dual high-specificity RGNs with optimized components were investigated for permanently repairing defective DMD alleles either through exon 51-targeted indel formation or major mutational hotspot excision (>500 kb), respectively. Firstly, we establish that, at high doses, third-generation HC-AdVs lacking all viral genes are significantly less cytotoxic than second-generation adenoviral vectors deleted in E1 and E2A. Secondly, we demonstrate that genetically retargeted HC-AdVs can correct up to 42% ± 13% of defective DMD alleles in muscle cell populations through targeted removal of the major mutational hotspot, in which over 60% of frame-shifting large deletions locate. Both DMD gene repair strategies tested readily led to the detection of Becker-like dystrophins in unselected muscle cell populations, leading to the restoration of β-dystroglycan at the plasmalemma of differentiated muscle cells. Hence, HC-AdVs permit the effective assessment of DMD gene-editing tools and strategies in dystrophin-defective human cells while broadening the gamut of DMD-correcting agents. Full article
(This article belongs to the Special Issue CRISPR Genome Editing)
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Review

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Open AccessFeature PaperReview
Improving Precise CRISPR Genome Editing by Small Molecules: Is there a Magic Potion?
Cells 2020, 9(5), 1318; https://doi.org/10.3390/cells9051318 - 25 May 2020
Abstract
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing has become a standard method in molecular biology, for the establishment of genetically modified cellular and animal models, for the identification and validation of drug targets in animals, and is heavily tested for use [...] Read more.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing has become a standard method in molecular biology, for the establishment of genetically modified cellular and animal models, for the identification and validation of drug targets in animals, and is heavily tested for use in gene therapy of humans. While the efficiency of CRISPR mediated gene targeting is much higher than of classical targeted mutagenesis, the efficiency of CRISPR genome editing to introduce defined changes into the genome is still low. Overcoming this problem will have a great impact on the use of CRISPR genome editing in academic and industrial research and the clinic. This review will present efforts to achieve this goal by small molecules, which modify the DNA repair mechanisms to facilitate the precise alteration of the genome. Full article
(This article belongs to the Special Issue CRISPR Genome Editing)
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Open AccessReview
Computational Tools and Resources Supporting CRISPR-Cas Experiments
Cells 2020, 9(5), 1288; https://doi.org/10.3390/cells9051288 - 22 May 2020
Abstract
The CRISPR-Cas system has become a cutting-edge technology that revolutionized genome engineering. The use of Cas9 nuclease is currently the method of choice in most tasks requiring a specific DNA modification. The rapid development in the field of CRISPR-Cas is reflected by the [...] Read more.
The CRISPR-Cas system has become a cutting-edge technology that revolutionized genome engineering. The use of Cas9 nuclease is currently the method of choice in most tasks requiring a specific DNA modification. The rapid development in the field of CRISPR-Cas is reflected by the constantly expanding ecosystem of computational tools aimed at facilitating experimental design and result analysis. The first group of CRISPR-Cas-related tools that we review is dedicated to aid in guide RNA design by prediction of their efficiency and specificity. The second, relatively new group of tools exploits the observed biases in repair outcomes to predict the results of CRISPR-Cas edits. The third class of tools is developed to assist in the evaluation of the editing outcomes by analysis of the sequencing data. These utilities are accompanied by relevant repositories and databases. Here we present a comprehensive and updated overview of the currently available CRISPR-Cas-related tools, from the perspective of a user who needs a convenient and reliable means to facilitate genome editing experiments at every step, from the guide RNA design to analysis of editing outcomes. Moreover, we discuss the current limitations and challenges that the field must overcome for further improvement in the CRISPR-Cas endeavor. Full article
(This article belongs to the Special Issue CRISPR Genome Editing)
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Open AccessReview
CRISPR/Cas9 Epigenome Editing Potential for Rare Imprinting Diseases: A Review
Cells 2020, 9(4), 993; https://doi.org/10.3390/cells9040993 - 16 Apr 2020
Abstract
Imprinting diseases (IDs) are rare congenital disorders caused by aberrant dosages of imprinted genes. Rare IDs are comprised by a group of several distinct disorders that share a great deal of homology in terms of genetic etiologies and symptoms. Disruption of genetic or [...] Read more.
Imprinting diseases (IDs) are rare congenital disorders caused by aberrant dosages of imprinted genes. Rare IDs are comprised by a group of several distinct disorders that share a great deal of homology in terms of genetic etiologies and symptoms. Disruption of genetic or epigenetic mechanisms can cause issues with regulating the expression of imprinted genes, thus leading to disease. Genetic mutations affect the imprinted genes, duplications, deletions, and uniparental disomy (UPD) are reoccurring phenomena causing imprinting diseases. Epigenetic alterations on methylation marks in imprinting control centers (ICRs) also alters the expression patterns and the majority of patients with rare IDs carries intact but either silenced or overexpressed imprinted genes. Canonical CRISPR/Cas9 editing relying on double-stranded DNA break repair has little to offer in terms of therapeutics for rare IDs. Instead CRISPR/Cas9 can be used in a more sophisticated way by targeting the epigenome. Catalytically dead Cas9 (dCas9) tethered with effector enzymes such as DNA de- and methyltransferases and histone code editors in addition to systems such as CRISPRa and CRISPRi have been shown to have high epigenome editing efficiency in eukaryotic cells. This new era of CRISPR epigenome editors could arguably be a game-changer for curing and treating rare IDs by refined activation and silencing of disturbed imprinted gene expression. This review describes major CRISPR-based epigenome editors and points out their potential use in research and therapy of rare imprinting diseases. Full article
(This article belongs to the Special Issue CRISPR Genome Editing)
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Open AccessReview
Adenoviral Vectors Meet Gene Editing: A Rising Partnership for the Genomic Engineering of Human Stem Cells and Their Progeny
Cells 2020, 9(4), 953; https://doi.org/10.3390/cells9040953 - 13 Apr 2020
Abstract
Gene editing permits changing specific DNA sequences within the vast genomes of human cells. Stem cells are particularly attractive targets for gene editing interventions as their self-renewal and differentiation capabilities consent studying cellular differentiation processes, screening small-molecule drugs, modeling human disorders, and testing [...] Read more.
Gene editing permits changing specific DNA sequences within the vast genomes of human cells. Stem cells are particularly attractive targets for gene editing interventions as their self-renewal and differentiation capabilities consent studying cellular differentiation processes, screening small-molecule drugs, modeling human disorders, and testing regenerative medicines. To integrate gene editing and stem cell technologies, there is a critical need for achieving efficient delivery of the necessary molecular tools in the form of programmable DNA-targeting enzymes and/or exogenous nucleic acid templates. Moreover, the impact that the delivery agents themselves have on the performance and precision of gene editing procedures is yet another critical parameter to consider. Viral vectors consisting of recombinant replication-defective viruses are under intense investigation for bringing about efficient gene-editing tool delivery and precise gene-editing in human cells. In this review, we focus on the growing role that adenoviral vectors are playing in the targeted genetic manipulation of human stem cells, progenitor cells, and their differentiated progenies in the context of in vitro and ex vivo protocols. As preamble, we provide an overview on the main gene editing principles and adenoviral vector platforms and end by discussing the possibilities ahead resulting from leveraging adenoviral vector, gene editing, and stem cell technologies. Full article
(This article belongs to the Special Issue CRISPR Genome Editing)
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