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CRISPR-Cas: Interactions with Genome and Physiological Maintenance
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CRISPR-Cas: Interactions with Genome and Physiological Maintenance

A special issue of Genes (ISSN 2073-4425). This special issue belongs to the section "Molecular Genetics and Genomics".

Deadline for manuscript submissions: closed (16 November 2020) | Viewed by 44695

Special Issue Editors

Dr. Ed Bolt

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Guest Editor
Queen's Medical Centre, The University of Nottingham Medical School, Nottingham NG7 2UH, UK
Interests: molecular biology; protein biochemistry; CRISPR-cas immunity; homologous recombination; DNA Repair
Dr. Christian Rudolph

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Guest Editor
Department of Life Sciences, Brunel University, London UB8 3PN, UK
Interests: cell biology; DNA replication; replication termination; chromosome dynamics
Dr. Ivana Ivancic-Bace

E-Mail Website
Guest Editor
Horvatovac 102a, Department of Biology, University of Zagreb, 10000 Zagreb, Croatia
Interests: genetic analysis; CRISPR-Cas immunity; homologous recombination; DNA repair

Special Issue Information

Dear Colleagues,

CRISPR-Cas enzymes provide a growing smorgasbord of tools for genetic engineering of DNA and RNA, and in genome editing to alter cell physiology in bacteria, plants and mammals. Interactions between CRISPR-Cas and host DNA repair enzymes are important for successful genome editing because editing enzymes generate DNA damage sites. These trigger DNA repair systems and can provoke wider genomic stress with potential to disrupt DNA replication and cell cycle progression. In their native cells CRISPR-Cas adaptive immunity systems functionally interact with DNA repair and genome stability systems, factors that promote building of the DNA-based CRISPR immunity system. CRISPR-Cas enzymes also impact on other physiological systems in interesting ways by mechanisms unknown, for example in bacterial biofilm formation. Understanding interplay between CRISPR-Cas enzymes and other host physiologies is a frontier for improving efficacy of gene-editing protocols, furthering understanding of DNA repair in healthcare, and for understanding prokaryotic biology. This collection of articles will evaluate current knowledge, for example:

  • Developments in understanding off-target effects of class II CRISPR-Cas systems; Cas9, Cas12a, Cas13a.
  • Interactions of DNA repair, replication and recombination with native CRISPR-Cas systems.
  • Interactions of DNA repair, replication and recombination with heterologous CRISPR-Cas systems used in genome editing.
  • Genome editing in plants.
  • Genome editing for growth, sustainability and development projects and in human disease.
  • DNA editing using adaptation enzymes Cas1-Cas2.
  • Class III CRISPR systems and cell signaling.
  • Class I CRISPR systems and biofilm formation.

Dr. Ed Bolt
Dr. Christian Rudolph
Dr. Ivana Ivancic-Bace
Guest Editors

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. Genes is an international peer-reviewed open access monthly 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 2600 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-Cas
  • DNA repair
  • genome editing
  • bacterial physiology
  • microbiology
  • homologous recombination
  • helicases
  • nucleases

Published Papers (7 papers)

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Research

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16 pages, 2251 KiB  
Article
Genome-Wide Analysis of Off-Target CRISPR/Cas9 Activity in Single-Cell-Derived Human Hematopoietic Stem and Progenitor Cell Clones
by Richard H. Smith, Yun-Ching Chen, Fayaz Seifuddin, Daniel Hupalo, Camille Alba, Robert Reger, Xin Tian, Daisuke Araki, Clifton L. Dalgard, Richard W. Childs, Mehdi Pirooznia and Andre Larochelle
Genes 2020, 11(12), 1501; https://doi.org/10.3390/genes11121501 - 13 Dec 2020
Cited by 14 | Viewed by 5131
Abstract
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated genome editing holds remarkable promise for the treatment of human genetic diseases. However, the possibility of off-target Cas9 activity remains a concern. To address this issue using clinically relevant target cells, we electroporated Cas9 [...] Read more.
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated genome editing holds remarkable promise for the treatment of human genetic diseases. However, the possibility of off-target Cas9 activity remains a concern. To address this issue using clinically relevant target cells, we electroporated Cas9 ribonucleoprotein (RNP) complexes (independently targeted to two different genomic loci, the CXCR4 locus on chromosome 2 and the AAVS1 locus on chromosome 19) into human mobilized peripheral blood-derived hematopoietic stem and progenitor cells (HSPCs) and assessed the acquisition of somatic mutations in an unbiased, genome-wide manner via whole genome sequencing (WGS) of single-cell-derived HSPC clones. Bioinformatic analysis identified >20,000 total somatic variants (indels, single nucleotide variants, and structural variants) distributed among Cas9-treated and non-Cas9-treated control HSPC clones. Statistical analysis revealed no significant difference in the number of novel non-targeted indels among the samples. Moreover, data analysis showed no evidence of Cas9-mediated indel formation at 623 predicted off-target sites. The median number of novel single nucleotide variants was slightly elevated in Cas9 RNP-recipient sample groups compared to baseline, but did not reach statistical significance. Structural variants were rare and demonstrated no clear causal connection to Cas9-mediated gene editing procedures. We find that the collective somatic mutational burden observed within Cas9 RNP-edited human HSPC clones is indistinguishable from naturally occurring levels of background genetic heterogeneity. Full article
(This article belongs to the Special Issue CRISPR-Cas: Interactions with Genome and Physiological Maintenance)
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15 pages, 2945 KiB  
Article
Genome Maintenance Proteins Modulate Autoimmunity Mediated Primed Adaptation by the Escherichia coli Type I-E CRISPR-Cas System
by Elena Kurilovich, Anna Shiriaeva, Anastasia Metlitskaya, Natalia Morozova, Ivana Ivancic-Bace, Konstantin Severinov and Ekaterina Savitskaya
Genes 2019, 10(11), 872; https://doi.org/10.3390/genes10110872 - 31 Oct 2019
Cited by 7 | Viewed by 4454
Abstract
Bacteria and archaea use CRISPR-Cas adaptive immunity systems to interfere with viruses, plasmids, and other mobile genetic elements. During the process of adaptation, CRISPR-Cas systems acquire immunity by incorporating short fragments of invaders’ genomes into CRISPR arrays. The acquisition of fragments of host [...] Read more.
Bacteria and archaea use CRISPR-Cas adaptive immunity systems to interfere with viruses, plasmids, and other mobile genetic elements. During the process of adaptation, CRISPR-Cas systems acquire immunity by incorporating short fragments of invaders’ genomes into CRISPR arrays. The acquisition of fragments of host genomes leads to autoimmunity and may drive chromosomal rearrangements, negative cell selection, and influence bacterial evolution. In this study, we investigated the role of proteins involved in genome stability maintenance in spacer acquisition by the Escherichia coli type I-E CRISPR-Cas system targeting its own genome. We show here, that the deletion of recJ decreases adaptation efficiency and affects accuracy of spacers incorporation into CRISPR array. Primed adaptation efficiency is also dramatically inhibited in double mutants lacking recB and sbcD but not in single mutants suggesting independent involvement and redundancy of RecBCD and SbcCD pathways in spacer acquisition. While the presence of at least one of two complexes is crucial for efficient primed adaptation, RecBCD and SbcCD affect the pattern of acquired spacers. Overall, our data suggest distinct roles of the RecBCD and SbcCD complexes and of RecJ in spacer precursor selection and insertion into CRISPR array and highlight the functional interplay between CRISPR-Cas systems and host genome maintenance mechanisms. Full article
(This article belongs to the Special Issue CRISPR-Cas: Interactions with Genome and Physiological Maintenance)
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14 pages, 2058 KiB  
Article
The Effect of DNA Topology on Observed Rates of R-Loop Formation and DNA Strand Cleavage by CRISPR Cas12a
by Kara van Aelst, Carlos J. Martínez-Santiago, Stephen J. Cross and Mark D. Szczelkun
Genes 2019, 10(2), 169; https://doi.org/10.3390/genes10020169 - 22 Feb 2019
Cited by 18 | Viewed by 5367
Abstract
Here we explored the mechanism of R-loop formation and DNA cleavage by type V CRISPR Cas12a (formerly known as Cpf1). We first used a single-molecule magnetic tweezers (MT) assay to show that R-loop formation by Lachnospiraceae bacterium ND2006 Cas12a is significantly enhanced by [...] Read more.
Here we explored the mechanism of R-loop formation and DNA cleavage by type V CRISPR Cas12a (formerly known as Cpf1). We first used a single-molecule magnetic tweezers (MT) assay to show that R-loop formation by Lachnospiraceae bacterium ND2006 Cas12a is significantly enhanced by negative DNA supercoiling, as observed previously with Streptococcus thermophilus DGCC7710 CRISPR3 Cas9. Consistent with the MT data, the apparent rate of cleavage of supercoiled plasmid DNA was observed to be >50-fold faster than the apparent rates for linear DNA or nicked circular DNA because of topology-dependent differences in R-loop formation kinetics. Taking the differences into account, the cleavage data for all substrates can be fitted with the same apparent rate constants for the two strand-cleavage steps, with the first event >15-fold faster than the second. By independently following the ensemble cleavage of the non-target strand (NTS) and target strand (TS), we could show that the faster rate is due to NTS cleavage, the slower rate due to TS cleavage, as expected from previous studies. Full article
(This article belongs to the Special Issue CRISPR-Cas: Interactions with Genome and Physiological Maintenance)
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Review

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18 pages, 3868 KiB  
Review
CRISPR/Cas9 in Cancer Immunotherapy: Animal Models and Human Clinical Trials
by Khalil Khalaf, Krzysztof Janowicz, Marta Dyszkiewicz-Konwińska, Greg Hutchings, Claudia Dompe, Lisa Moncrieff, Maurycy Jankowski, Marta Machnik, Urszula Oleksiewicz, Ievgeniia Kocherova, Jim Petitte, Paul Mozdziak, Jamil A. Shibli, Dariusz Iżycki, Małgorzata Józkowiak, Hanna Piotrowska-Kempisty, Mariusz T. Skowroński, Paweł Antosik and Bartosz Kempisty
Genes 2020, 11(8), 921; https://doi.org/10.3390/genes11080921 - 11 Aug 2020
Cited by 27 | Viewed by 10288
Abstract
Even though chemotherapy and immunotherapy emerged to limit continual and unregulated proliferation of cancer cells, currently available therapeutic agents are associated with high toxicity levels and low success rates. Additionally, ongoing multi-targeted therapies are limited only for few carcinogenesis pathways, due to continually [...] Read more.
Even though chemotherapy and immunotherapy emerged to limit continual and unregulated proliferation of cancer cells, currently available therapeutic agents are associated with high toxicity levels and low success rates. Additionally, ongoing multi-targeted therapies are limited only for few carcinogenesis pathways, due to continually emerging and evolving mutations of proto-oncogenes and tumor-suppressive genes. CRISPR/Cas9, as a specific gene-editing tool, is used to correct causative mutations with minimal toxicity, but is also employed as an adjuvant to immunotherapy to achieve a more robust immunological response. Some of the most critical limitations of the CRISPR/Cas9 technology include off-target mutations, resulting in nonspecific restrictions of DNA upstream of the Protospacer Adjacent Motifs (PAM), ethical agreements, and the lack of a scientific consensus aiming at risk evaluation. Currently, CRISPR/Cas9 is tested on animal models to enhance genome editing specificity and induce a stronger anti-tumor response. Moreover, ongoing clinical trials use the CRISPR/Cas9 system in immune cells to modify genomes in a target-specific manner. Recently, error-free in vitro systems have been engineered to overcome limitations of this gene-editing system. The aim of the article is to present the knowledge concerning the use of CRISPR Cas9 technique in targeting treatment-resistant cancers. Additionally, the use of CRISPR/Cas9 is aided as an emerging supplementation of immunotherapy, currently used in experimental oncology. Demonstrating further, applications and advances of the CRISPR/Cas9 technique are presented in animal models and human clinical trials. Concluding, an overview of the limitations of the gene-editing tool is proffered. Full article
(This article belongs to the Special Issue CRISPR-Cas: Interactions with Genome and Physiological Maintenance)
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17 pages, 2289 KiB  
Review
Therapeutic Editing of the TP53 Gene: Is CRISPR/Cas9 an Option?
by Regina Mirgayazova, Raniya Khadiullina, Vitaly Chasov, Rimma Mingaleeva, Regina Miftakhova, Albert Rizvanov and Emil Bulatov
Genes 2020, 11(6), 704; https://doi.org/10.3390/genes11060704 - 25 Jun 2020
Cited by 33 | Viewed by 7828
Abstract
The TP53 gene encodes the transcription factor and oncosuppressor p53 protein that regulates a multitude of intracellular metabolic pathways involved in DNA damage repair, cell cycle arrest, apoptosis, and senescence. In many cases, alterations (e.g., mutations of the TP53 gene) negatively affect these [...] Read more.
The TP53 gene encodes the transcription factor and oncosuppressor p53 protein that regulates a multitude of intracellular metabolic pathways involved in DNA damage repair, cell cycle arrest, apoptosis, and senescence. In many cases, alterations (e.g., mutations of the TP53 gene) negatively affect these pathways resulting in tumor development. Recent advances in genome manipulation technologies, CRISPR/Cas9, in particular, brought us closer to therapeutic gene editing for the treatment of cancer and hereditary diseases. Genome-editing therapies for blood disorders, blindness, and cancer are currently being evaluated in clinical trials. Eventually CRISPR/Cas9 technology is expected to target TP53 as the most mutated gene in all types of cancers. A majority of TP53 mutations are missense which brings immense opportunities for the CRISPR/Cas9 system that has been successfully used for correcting single nucleotides in various models, both in vitro and in vivo. In this review, we highlight the recent clinical applications of CRISPR/Cas9 technology for therapeutic genome editing and discuss its perspectives for editing TP53 and regulating transcription of p53 pathway genes. Full article
(This article belongs to the Special Issue CRISPR-Cas: Interactions with Genome and Physiological Maintenance)
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14 pages, 2496 KiB  
Review
Cas3 Protein—A Review of a Multi-Tasking Machine
by Liu He, Michael St. John James, Marin Radovcic, Ivana Ivancic-Bace and Edward L. Bolt
Genes 2020, 11(2), 208; https://doi.org/10.3390/genes11020208 - 18 Feb 2020
Cited by 18 | Viewed by 6370
Abstract
Cas3 has essential functions in CRISPR immunity but its other activities and roles, in vitro and in cells, are less widely known. We offer a concise review of the latest understanding and questions arising from studies of Cas3 mechanism during CRISPR immunity, and [...] Read more.
Cas3 has essential functions in CRISPR immunity but its other activities and roles, in vitro and in cells, are less widely known. We offer a concise review of the latest understanding and questions arising from studies of Cas3 mechanism during CRISPR immunity, and highlight recent attempts at using Cas3 for genetic editing. We then spotlight involvement of Cas3 in other aspects of cell biology, for which understanding is lacking—these focus on CRISPR systems as regulators of cellular processes in addition to defense against mobile genetic elements. Full article
(This article belongs to the Special Issue CRISPR-Cas: Interactions with Genome and Physiological Maintenance)
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Other

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9 pages, 1189 KiB  
Concept Paper
A Gold Standard, CRISPR/Cas9-Based Complementation Strategy Reliant on 24 Nucleotide Bookmark Sequences
by François M. Seys, Peter Rowe, Edward L. Bolt, Christopher M. Humphreys and Nigel P. Minton
Genes 2020, 11(4), 458; https://doi.org/10.3390/genes11040458 - 23 Apr 2020
Cited by 12 | Viewed by 4164
Abstract
Phenotypic complementation of gene knockouts is an essential step in establishing function. Here, we describe a simple strategy for ‘gold standard’ complementation in which the mutant allele is replaced in situ with a wild type (WT) allele in a procedure that exploits CRISPR/Cas9. [...] Read more.
Phenotypic complementation of gene knockouts is an essential step in establishing function. Here, we describe a simple strategy for ‘gold standard’ complementation in which the mutant allele is replaced in situ with a wild type (WT) allele in a procedure that exploits CRISPR/Cas9. The method relies on the prior incorporation of a unique 24 nucleotide (nt) ‘bookmark’ sequence into the mutant allele to act as a guide RNA target during its Cas9-mediated replacement with the WT allele. The bookmark comprises a 23 nt Cas9 target sequence plus an additional nt to ensure the deletion is in-frame. Here, bookmarks are tailored to Streptococcus pyogenes CRISPR/Cas9 but could be designed for any CRISPR/Cas system. For proof of concept, nine bookmarks were tested in Clostridium autoethanogenum. Complementation efficiencies reached 91%. As complemented strains are indistinguishable from their progenitors, concerns over contamination may be satisfied by the incorporation of ‘watermark’ sequences into the complementing genes. Full article
(This article belongs to the Special Issue CRISPR-Cas: Interactions with Genome and Physiological Maintenance)
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