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Cells
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CRISPR Genome Editing
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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) | Viewed by 84674

Special Issue Editor

Prof. Dr. Cord Brakebusch

E-Mail Website
Guest Editor
Biotech Research & Innovation Centre, The University of Copenhagen, Copenhagen, Denmark
Interests: Rho GTPases; keratinocytes; mouse disease models
Special Issues, Collections and Topics 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

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Cells is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

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

Related Special Issue

Published Papers (11 papers)

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Editorial

Jump to: Research, Review

2 pages, 172 KiB  
Editorial
CRISPR Genome Editing: How to Make a Fantastic Method Even Better
by Cord Brakebusch
Cells 2021, 10(2), 408; https://0-doi-org.brum.beds.ac.uk/10.3390/cells10020408 - 16 Feb 2021
Cited by 4 | Viewed by 2243
Abstract
CRISPR genome editing describes targeted mutagenesis involving a programmable DNA scissor consisting of a protein (Cas9) bound to a short RNA [...] Full article
(This article belongs to the Special Issue CRISPR Genome Editing)

Research

Jump to: Editorial, Review

22 pages, 8655 KiB  
Article
Using High-Content Screening to Generate Single-Cell Gene-Corrected Patient-Derived iPS Clones Reveals Excess Alpha-Synuclein with Familial Parkinson’s Disease Point Mutation A30P
by Peter Barbuti, Paul Antony, Bruno Santos, François Massart, Gérald Cruciani, Claire Dording, Jonathan Arias, Jens Schwamborn and Rejko Krüger
Cells 2020, 9(9), 2065; https://0-doi-org.brum.beds.ac.uk/10.3390/cells9092065 - 10 Sep 2020
Cited by 14 | Viewed by 4552
Abstract
The generation of isogenic induced pluripotent stem cell (iPSC) lines using CRISPR-Cas9 technology is a technically challenging, time-consuming process with variable efficiency. Here we use fluorescence-activated cell sorting (FACS) to sort biallelic CRISPR-Cas9 edited single-cell iPSC clones into high-throughput 96-well microtiter plates. We [...] Read more.
The generation of isogenic induced pluripotent stem cell (iPSC) lines using CRISPR-Cas9 technology is a technically challenging, time-consuming process with variable efficiency. Here we use fluorescence-activated cell sorting (FACS) to sort biallelic CRISPR-Cas9 edited single-cell iPSC clones into high-throughput 96-well microtiter plates. We used high-content screening (HCS) technology and generated an in-house developed algorithm to select the correctly edited isogenic clones for continued expansion and validation. In our model we have gene-corrected the iPSCs of a Parkinson’s disease (PD) patient carrying the autosomal dominantly inherited heterozygous c.88G>C mutation in the SNCA gene, which leads to the pathogenic p.A30P form of the alpha-synuclein protein. Undertaking a PCR restriction-digest mediated clonal selection strategy prior to sequencing, we were able to post-sort validate each isogenic clone using a quadruple screening strategy prior to generating footprint-free isogenic iPSC lines, retaining a normal molecular karyotype, pluripotency and three germ-layer differentiation potential. Directed differentiation into midbrain dopaminergic neurons revealed that SNCA expression is reduced in the gene-corrected clones, which was validated by a reduction at the alpha-synuclein protein level. The generation of single-cell isogenic clones facilitates new insights in the role of alpha-synuclein in PD and furthermore is applicable across patient-derived disease models. Full article
(This article belongs to the Special Issue CRISPR Genome Editing)
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13 pages, 1324 KiB  
Article
BE4max and AncBE4max Are Efficient in Germline Conversion of C:G to T:A Base Pairs in Zebrafish
by Blake Carrington, Rachel N. Weinstein and Raman Sood
Cells 2020, 9(7), 1690; https://0-doi-org.brum.beds.ac.uk/10.3390/cells9071690 - 14 Jul 2020
Cited by 14 | Viewed by 4127
Abstract
The ease of use and robustness of genome editing by CRISPR/Cas9 has led to successful use of gene knockout zebrafish for disease modeling. However, it still remains a challenge to precisely edit the zebrafish genome to create single-nucleotide substitutions, which account for ~60% [...] Read more.
The ease of use and robustness of genome editing by CRISPR/Cas9 has led to successful use of gene knockout zebrafish for disease modeling. However, it still remains a challenge to precisely edit the zebrafish genome to create single-nucleotide substitutions, which account for ~60% of human disease-causing mutations. Recently developed base editing nucleases provide an excellent alternate to CRISPR/Cas9-mediated homology dependent repair for generation of zebrafish with point mutations. A new set of cytosine base editors, termed BE4max and AncBE4max, demonstrated improved base editing efficiency in mammalian cells but have not been evaluated in zebrafish. Therefore, we undertook this study to evaluate their efficiency in converting C:G to T:A base pairs in zebrafish by somatic and germline analysis using highly active sgRNAs to twist and ntl genes. Our data demonstrated that these improved BE4max set of plasmids provide desired base substitutions at similar efficiency and without any indels compared to the previously reported BE3 and Target-AID plasmids in zebrafish. Our data also showed that AncBE4max produces fewer incorrect and bystander edits, suggesting that it can be further improved by codon optimization of its components for use in zebrafish. Full article
(This article belongs to the Special Issue CRISPR Genome Editing)
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17 pages, 2409 KiB  
Article
Development of Cellular Models to Study Efficiency and Safety of Gene Edition by Homologous Directed Recombination Using the CRISPR/Cas9 System
by Sabina Sánchez-Hernández, Araceli Aguilar-González, Beatriz Guijarro-Albaladejo, Noelia Maldonado-Pérez, Iris Ramos-Hernández, Marina Cortijo-Gutiérrez, Rosario María Sánchez Martín, Karim Benabdellah and Francisco Martin
Cells 2020, 9(6), 1492; https://0-doi-org.brum.beds.ac.uk/10.3390/cells9061492 - 18 Jun 2020
Cited by 1 | Viewed by 4175
Abstract
In spite of the enormous potential of CRISPR/Cas in basic and applied science, the levels of undesired genomic modifications cells still remain mostly unknown and controversial. Nowadays, the efficiency and specificity of the cuts generated by CRISPR/Cas is the main concern. However, there [...] Read more.
In spite of the enormous potential of CRISPR/Cas in basic and applied science, the levels of undesired genomic modifications cells still remain mostly unknown and controversial. Nowadays, the efficiency and specificity of the cuts generated by CRISPR/Cas is the main concern. However, there are also other potential drawbacks when DNA donors are used for gene repair or gene knock-ins. These GE strategies should take into account not only the specificity of the nucleases, but also the fidelity of the DNA donor to carry out their function. The current methods to quantify the fidelity of DNA donor are costly and lack sensitivity to detect illegitimate DNA donor integrations. In this work, we have engineered two reporter cell lines (K562_SEWAS84 and K562GWP) that efficiently quantify both the on-target and the illegitimate DNA donor integrations in a WAS-locus targeting setting. K562_SEWAS84 cells allow the detection of both HDR-and HITI-based donor integration, while K562GWP cells only report HDR-based GE. To the best of our knowledge, these are the first reporter systems that allow the use of gRNAs targeting a relevant locus to measure efficacy and specificity of DNA donor-based GE strategies. By using these models, we have found that the specificity of HDR is independent of the delivery method and that the insertion of the target sequence into the DNA donor enhances efficiency but do not affect specificity. Finally, we have also shown that the higher the number of the target sites is, the higher the specificity and efficacy of GE will be. Full article
(This article belongs to the Special Issue CRISPR Genome Editing)
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24 pages, 12145 KiB  
Article
High-Capacity Adenoviral Vectors Permit Robust and Versatile Testing of DMD Gene Repair Tools and Strategies in Human Cells
by Marcella Brescia, Josephine M. Janssen, Jin Liu and Manuel A. F. V. Gonçalves
Cells 2020, 9(4), 869; https://0-doi-org.brum.beds.ac.uk/10.3390/cells9040869 - 02 Apr 2020
Cited by 19 | Viewed by 3979
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

Jump to: Editorial, Research

21 pages, 2153 KiB  
Review
DNA Damage: From Threat to Treatment
by Antonio Carusillo and Claudio Mussolino
Cells 2020, 9(7), 1665; https://0-doi-org.brum.beds.ac.uk/10.3390/cells9071665 - 10 Jul 2020
Cited by 99 | Viewed by 10047
Abstract
DNA is the source of genetic information, and preserving its integrity is essential in order to sustain life. The genome is continuously threatened by different types of DNA lesions, such as abasic sites, mismatches, interstrand crosslinks, or single-stranded and double-stranded breaks. As a [...] Read more.
DNA is the source of genetic information, and preserving its integrity is essential in order to sustain life. The genome is continuously threatened by different types of DNA lesions, such as abasic sites, mismatches, interstrand crosslinks, or single-stranded and double-stranded breaks. As a consequence, cells have evolved specialized DNA damage response (DDR) mechanisms to sustain genome integrity. By orchestrating multilayer signaling cascades specific for the type of lesion that occurred, the DDR ensures that genetic information is preserved overtime. In the last decades, DNA repair mechanisms have been thoroughly investigated to untangle these complex networks of pathways and processes. As a result, key factors have been identified that control and coordinate DDR circuits in time and space. In the first part of this review, we describe the critical processes encompassing DNA damage sensing and resolution. In the second part, we illustrate the consequences of partial or complete failure of the DNA repair machinery. Lastly, we will report examples in which this knowledge has been instrumental to develop novel therapies based on genome editing technologies, such as CRISPR-Cas. Full article
(This article belongs to the Special Issue CRISPR Genome Editing)
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25 pages, 1578 KiB  
Review
Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing
by Muhammad Naeem, Saman Majeed, Mubasher Zahir Hoque and Irshad Ahmad
Cells 2020, 9(7), 1608; https://0-doi-org.brum.beds.ac.uk/10.3390/cells9071608 - 02 Jul 2020
Cited by 232 | Viewed by 25065
Abstract
Gene editing that makes target gene modification in the genome by deletion or addition has revolutionized the era of biomedicine. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 emerged as a substantial tool due to its simplicity in use, less cost and extraordinary efficiency [...] Read more.
Gene editing that makes target gene modification in the genome by deletion or addition has revolutionized the era of biomedicine. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 emerged as a substantial tool due to its simplicity in use, less cost and extraordinary efficiency than the conventional gene-editing tools, including zinc finger nucleases (ZFNs) and Transcription activator-like effector nucleases (TALENs). However, potential off-target activities are crucial shortcomings in the CRISPR system. Numerous types of approaches have been developed to reduce off-target effects. Here, we review several latest approaches to reduce the off-target effects, including biased or unbiased off-target detection, cytosine or adenine base editors, prime editing, dCas9, Cas9 paired nickase, ribonucleoprotein (RNP) delivery and truncated gRNAs. This review article provides extensive information to cautiously interpret off-target effects to assist the basic and clinical applications in biomedicine. Full article
(This article belongs to the Special Issue CRISPR Genome Editing)
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16 pages, 885 KiB  
Review
Improving Precise CRISPR Genome Editing by Small Molecules: Is there a Magic Potion?
by Nadja Bischoff, Sandra Wimberger, Marcello Maresca and Cord Brakebusch
Cells 2020, 9(5), 1318; https://0-doi-org.brum.beds.ac.uk/10.3390/cells9051318 - 25 May 2020
Cited by 37 | Viewed by 6149
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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17 pages, 1385 KiB  
Review
Computational Tools and Resources Supporting CRISPR-Cas Experiments
by Pawel Sledzinski, Mateusz Nowaczyk and Marta Olejniczak
Cells 2020, 9(5), 1288; https://0-doi-org.brum.beds.ac.uk/10.3390/cells9051288 - 22 May 2020
Cited by 34 | Viewed by 6622
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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25 pages, 3582 KiB  
Review
CRISPR/Cas9 Epigenome Editing Potential for Rare Imprinting Diseases: A Review
by Linn Amanda Syding, Petr Nickl, Petr Kasparek and Radislav Sedlacek
Cells 2020, 9(4), 993; https://0-doi-org.brum.beds.ac.uk/10.3390/cells9040993 - 16 Apr 2020
Cited by 32 | Viewed by 9648
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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28 pages, 4131 KiB  
Review
Adenoviral Vectors Meet Gene Editing: A Rising Partnership for the Genomic Engineering of Human Stem Cells and Their Progeny
by Francesca Tasca, Qian Wang and Manuel A.F.V. Gonçalves
Cells 2020, 9(4), 953; https://0-doi-org.brum.beds.ac.uk/10.3390/cells9040953 - 13 Apr 2020
Cited by 19 | Viewed by 5802
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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