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Cells
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Genome Editing Systems, Methods, Techniques and Their Application Series 2
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Genome Editing Systems, Methods, Techniques and Their Application Series 2

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

Deadline for manuscript submissions: closed (31 March 2022) | Viewed by 52044

Special Issue Editor

Dr. Tetsushi Sakuma

E-Mail Website
Guest Editor
Division of Integrated Sciences for Life, Graduate School of Integrated Sciences for Life, Hiroshima University, Hiroshima 739-8526, Japan
Interests: genome editing; epigenome editing; CRISPR-Cas9; TALEN; ZFN
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

With the rapid evolution of site-specific nuclease systems, such as CRISPR-Cas9 and TALEN, genome editing technology has led to a big breakthrough in the field of life science. Precision gene engineering has been achieved in various cells, animals, plants, and microorganisms, and the derivative technologies have been developed and applied in various ways, including transcriptional control, epigenome editing, chromosome visualization, genome-wide screening, and DNA barcoding.

This Special Issue covers original research and review papers involved in this type of genome editing and related technologies. A wide range of research topics is acceptable, such as the basic development of genome editing tools and methods, functional genomics studies, and biomedical applications using CRISPR-Cas9.

Prof. Dr. Tetsushi Sakuma
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

  • Genome editing
  • Gene knockout
  • Gene knock-in
  • Epigenome editing
  • CRISPR-Cas9
  • Transcription activator-like effector nuclease (TALEN)
  • Zinc-finger nuclease (ZFN)

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Published Papers (12 papers)

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Research

Jump to: Review

28 pages, 4759 KiB  
Article
The Bibliometric Landscape of Gene Editing Innovation and Regulation in the Worldwide
by Xun Wei, Aqing Pu, Qianqian Liu, Quancan Hou, Yong Zhang, Xueli An, Yan Long, Yilin Jiang, Zhenying Dong, Suowei Wu and Xiangyuan Wan
Cells 2022, 11(17), 2682; https://0-doi-org.brum.beds.ac.uk/10.3390/cells11172682 - 29 Aug 2022
Cited by 17 | Viewed by 3581
Abstract
Gene editing (GE) has become one of the mainstream bioengineering technologies over the past two decades, mainly fueled by the rapid development of the CRISPR/Cas system since 2012. To date, plenty of articles related to the progress and applications of GE have been [...] Read more.
Gene editing (GE) has become one of the mainstream bioengineering technologies over the past two decades, mainly fueled by the rapid development of the CRISPR/Cas system since 2012. To date, plenty of articles related to the progress and applications of GE have been published globally, but the objective, quantitative and comprehensive investigations of them are relatively few. Here, 13,980 research articles and reviews published since 1999 were collected by using GE-related queries in the Web of Science. We used bibliometric analysis to investigate the competitiveness and cooperation of leading countries, influential affiliations, and prolific authors. Text clustering methods were used to assess technical trends and research hotspots dynamically. The global application status and regulatory framework were also summarized. This analysis illustrates the bottleneck of the GE innovation and provides insights into the future trajectory of development and application of the technology in various fields, which will be helpful for the popularization of gene editing technology. Full article
(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application Series 2)
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13 pages, 1341 KiB  
Article
Homologous Recombination-Enhancing Factors Identified by Comparative Transcriptomic Analyses of Pluripotent Stem Cell of Human and Common Marmoset
by Sho Yoshimatsu, Mayutaka Nakajima, Emi Qian, Tsukasa Sanosaka, Tsukika Sato and Hideyuki Okano
Cells 2022, 11(3), 360; https://0-doi-org.brum.beds.ac.uk/10.3390/cells11030360 - 21 Jan 2022
Cited by 2 | Viewed by 2138
Abstract
A previous study assessing the efficiency of the genome editing technology CRISPR-Cas9 for knock-in gene targeting in common marmoset (marmoset; Callithrix jacchus) embryonic stem cells (ESCs) unexpectedly identified innately enhanced homologous recombination activity in marmoset ESCs. Here, we compared gene expression in marmoset [...] Read more.
A previous study assessing the efficiency of the genome editing technology CRISPR-Cas9 for knock-in gene targeting in common marmoset (marmoset; Callithrix jacchus) embryonic stem cells (ESCs) unexpectedly identified innately enhanced homologous recombination activity in marmoset ESCs. Here, we compared gene expression in marmoset and human pluripotent stem cells using transcriptomic and quantitative PCR analyses and found that five HR-related genes (BRCA1, BRCA2, RAD51C, RAD51D, and RAD51) were upregulated in marmoset cells. A total of four of these upregulated genes enhanced HR efficiency with CRISPR-Cas9 in human pluripotent stem cells. Thus, the present study provides a novel insight into species-specific mechanisms for the choice of DNA repair pathways. Full article
(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application Series 2)
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18 pages, 1795 KiB  
Article
Assessment of the Level of Accumulation of the dIFN Protein Integrated by the Knock-In Method into the Region of the Histone H3.3 Gene of Arabidopsis thaliana
by Natalya V. Permyakova, Tatyana V. Marenkova, Pavel A. Belavin, Alla A. Zagorskaya, Yuriy V. Sidorchuk, Elena A. Uvarova, Vitaliy V. Kuznetsov, Sergey M. Rozov and Elena V. Deineko
Cells 2021, 10(8), 2137; https://0-doi-org.brum.beds.ac.uk/10.3390/cells10082137 - 19 Aug 2021
Cited by 6 | Viewed by 1916
Abstract
Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We investigated the possibility of obtaining a suspension cell culture of Arabidopsis thaliana carrying a site-specific integration of [...] Read more.
Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We investigated the possibility of obtaining a suspension cell culture of Arabidopsis thaliana carrying a site-specific integration of a target gene encoding modified human interferon (dIFN) using endonuclease Cas9. For the targeted insertion, we selected the region of the histone H3.3 gene (HTR5) with a high constitutive level of expression. Our results indicated that Cas9-induced DNA integration occurred with the highest frequency with the construction with donor DNA surrounded by homology arms and Cas9 endonuclease recognition sites. Among the monoclones of the four cell lines with knock-in studied, there is high heterogeneity in the level of expression and accumulation of the target protein. The accumulation of dIFN protein in cell lines with targeted insertions into the target region of the HTR5 gene does not statistically differ from the level of accumulation of dIFN protein in the group of lines with random integration of the transgene. However, one among the monoclonal lines with knock-in has a dIFN accumulation level above 2% of TSP, which is very high. Full article
(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application Series 2)
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17 pages, 2115 KiB  
Article
Genome Editing to Generate Sake Yeast Strains with Eight Mutations That Confer Excellent Brewing Characteristics
by Tomoya Chadani, Shinsuke Ohnuki, Atsuko Isogai, Tetsuya Goshima, Mao Kashima, Farzan Ghanegolmohammadi, Tomoyuki Nishi, Dai Hirata, Daisuke Watanabe, Katsuhiko Kitamoto, Takeshi Akao and Yoshikazu Ohya
Cells 2021, 10(6), 1299; https://0-doi-org.brum.beds.ac.uk/10.3390/cells10061299 - 24 May 2021
Cited by 16 | Viewed by 3917
Abstract
Sake yeast is mostly diploid, so the introduction of recessive mutations to improve brewing characteristics requires considerable effort. To construct sake yeast with multiple excellent brewing characteristics, we used an evidence-based approach that exploits genome editing technology. Our breeding targeted the AWA1, [...] Read more.
Sake yeast is mostly diploid, so the introduction of recessive mutations to improve brewing characteristics requires considerable effort. To construct sake yeast with multiple excellent brewing characteristics, we used an evidence-based approach that exploits genome editing technology. Our breeding targeted the AWA1, CAR1, MDE1, and FAS2 genes. We introduced eight mutations into standard sake yeast to construct a non-foam-forming strain that makes sake without producing carcinogens or an unpleasant odor, while producing a sweet ginjo aroma. Small-scale fermentation tests showed that the desired sake could be brewed with our genome-edited strains. The existence of a few unexpected genetic perturbations introduced during breeding proved that genome editing technology is extremely effective for the serial breeding of sake yeast. Full article
(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application Series 2)
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15 pages, 1977 KiB  
Article
An Optimized Preparation Method for Long ssDNA Donors to Facilitate Quick Knock-In Mouse Generation
by Yukiko U. Inoue, Yuki Morimoto, Mayumi Yamada, Ryosuke Kaneko, Kazumi Shimaoka, Shinji Oki, Mayuko Hotta, Junko Asami, Eriko Koike, Kei Hori, Mikio Hoshino, Itaru Imayoshi and Takayoshi Inoue
Cells 2021, 10(5), 1076; https://0-doi-org.brum.beds.ac.uk/10.3390/cells10051076 - 30 Apr 2021
Cited by 7 | Viewed by 5863
Abstract
Fluorescent reporter mouse lines and Cre/Flp recombinase driver lines play essential roles in investigating various molecular functions in vivo. Now that applications of the CRISPR/Cas9 genome-editing system to mouse fertilized eggs have drastically accelerated these knock-in mouse generations, the next need is [...] Read more.
Fluorescent reporter mouse lines and Cre/Flp recombinase driver lines play essential roles in investigating various molecular functions in vivo. Now that applications of the CRISPR/Cas9 genome-editing system to mouse fertilized eggs have drastically accelerated these knock-in mouse generations, the next need is to establish easier, quicker, and cheaper methods for knock-in donor preparation. Here, we reverify and optimize the phospho-PCR method to obtain highly pure long single-stranded DNAs (ssDNAs) suitable for knock-in mouse generation via genome editing. The sophisticated sequential use of two exonucleases, in which double-stranded DNAs (dsDNAs) amplified by a pair of 5′-phosphorylated primer and normal primer are digested by Lambda exonuclease to yield ssDNA and the following Exonuclease III treatment degrades the remaining dsDNAs, enables much easier long ssDNA productions without laborious gel extraction steps. By microinjecting these donor DNAs along with CRISPR/Cas9 components into mouse zygotes, we have effectively generated fluorescent reporter lines and recombinase drivers. To further broaden the applicability, we have prepared long ssDNA donors in higher concentrations and electroporated them into mouse eggs to successfully obtain knock-in embryos. This classical yet improved method, which is regaining attention on the progress of CRISPR/Cas9 development, shall be the first choice for long donor DNA preparation, and the resulting knock-in lines could accelerate life science research. Full article
(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application Series 2)
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16 pages, 4751 KiB  
Article
Dynamic High-Sensitivity Quantitation of Procollagen-I by Endogenous CRISPR-Cas9 NanoLuciferase Tagging
by Ben C. Calverley, Karl E. Kadler and Adam Pickard
Cells 2020, 9(9), 2070; https://0-doi-org.brum.beds.ac.uk/10.3390/cells9092070 - 10 Sep 2020
Cited by 7 | Viewed by 3316
Abstract
The ability to quantitate a protein of interest temporally and spatially at subcellular resolution in living cells would generate new opportunities for research and drug discovery, but remains a major technical challenge. Here, we describe dynamic, high-sensitivity protein quantitation technique using NanoLuciferase (NLuc) [...] Read more.
The ability to quantitate a protein of interest temporally and spatially at subcellular resolution in living cells would generate new opportunities for research and drug discovery, but remains a major technical challenge. Here, we describe dynamic, high-sensitivity protein quantitation technique using NanoLuciferase (NLuc) tagging, which is effective across microscopy and multiwell platforms. Using collagen as a test protein, the CRISPR-Cas9-mediated introduction of nluc (encoding NLuc) into the Col1a2 locus enabled the simplification and miniaturisation of procollagen-I (PC-I) quantitation. Collagen was chosen because of the clinical interest in its dysregulation in cardiovascular and musculoskeletal disorders, and in fibrosis, which is a confounding factor in 45% of deaths, including those brought about by cancer. Collagen is also the cargo protein of choice for studying protein secretion because of its unusual shape and size. However, the use of overexpression promoters (which drowns out endogenous regulatory mechanisms) is often needed to achieve good signal/noise ratios in fluorescence microscopy of tagged collagen. We show that endogenous knock-in of NLuc, combined with its high brightness, negates the need to use exogenous promoters, preserves the circadian regulation of collagen synthesis and the responsiveness to TGF-β, and enables time-lapse microscopy of intracellular transport compartments containing procollagen cargo. In conclusion, we demonstrate the utility of CRISPR-Cas9-mediated endogenous NLuc tagging to robustly quantitate extracellular, intracellular, and subcellular protein levels and localisation. Full article
(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application Series 2)
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14 pages, 1765 KiB  
Communication
Hydrodynamics-Based Transplacental Delivery as a Useful Noninvasive Tool for Manipulating Fetal Genome
by Shingo Nakamura, Naoko Ando, Satoshi Watanabe, Eri Akasaka, Masayuki Ishihara and Masahiro Sato
Cells 2020, 9(7), 1744; https://0-doi-org.brum.beds.ac.uk/10.3390/cells9071744 - 21 Jul 2020
Cited by 6 | Viewed by 2309
Abstract
We previously demonstrated that the injection of pregnant wild-type female mice (carrying enhanced green fluorescent protein (EGFP)-expressing transgenic fetuses) at embryonic day (E) 12.5 with an all-in-one plasmid conferring the expression of both Cas9 and guide RNA (targeted to the EGFP cDNA) complexed [...] Read more.
We previously demonstrated that the injection of pregnant wild-type female mice (carrying enhanced green fluorescent protein (EGFP)-expressing transgenic fetuses) at embryonic day (E) 12.5 with an all-in-one plasmid conferring the expression of both Cas9 and guide RNA (targeted to the EGFP cDNA) complexed with the gene delivery reagent, resulted in some fetuses exhibiting reduced fluorescence in their hearts and gene insertion/deletion (indel) mutations. In this study, we examined whether the endogenous myosin heavy-chain α (MHCα) gene can be successfully genome-edited by this method in the absence of a gene delivery reagent with potential fetal toxicity. For this, we employed a hydrodynamics-based gene delivery (HGD) system with the aim of ensuring fetal gene delivery rates and biosafety. We also investigated which embryonic stages are suitable for the induction of genome editing in fetuses. Of the three pregnant females injected at E9.5, one had mutated fetuses: all examined fetuses carried exogenous plasmid DNA, and four of 10 (40%) exhibited mosaic indel mutations in MHCα. Gene delivery to fetuses at E12.5 and E15.5 did not cause mutations. Thus, the HGD-based transplacental delivery of a genome editing vector may be able to manipulate the fetal genomes of E9.5 fetuses. Full article
(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application Series 2)
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Review

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30 pages, 13578 KiB  
Review
Genetic Kidney Diseases (GKDs) Modeling Using Genome Editing Technologies
by Fernando Gómez-García, Raquel Martínez-Pulleiro, Noa Carrera, Catarina Allegue and Miguel A. Garcia-Gonzalez
Cells 2022, 11(9), 1571; https://0-doi-org.brum.beds.ac.uk/10.3390/cells11091571 - 06 May 2022
Cited by 2 | Viewed by 3873
Abstract
Genetic kidney diseases (GKDs) are a group of rare diseases, affecting approximately about 60 to 80 per 100,000 individuals, for which there is currently no treatment that can cure them (in many cases). GKDs usually leads to early-onset chronic kidney disease, which results [...] Read more.
Genetic kidney diseases (GKDs) are a group of rare diseases, affecting approximately about 60 to 80 per 100,000 individuals, for which there is currently no treatment that can cure them (in many cases). GKDs usually leads to early-onset chronic kidney disease, which results in patients having to undergo dialysis or kidney transplant. Here, we briefly describe genetic causes and phenotypic effects of six GKDs representative of different ranges of prevalence and renal involvement (ciliopathy, glomerulopathy, and tubulopathy). One of the shared characteristics of GKDs is that most of them are monogenic. This characteristic makes it possible to use site-specific nuclease systems to edit the genes that cause GKDs and generate in vitro and in vivo models that reflect the genetic abnormalities of GKDs. We describe and compare these site-specific nuclease systems (zinc finger nucleases (ZFNs), transcription activator-like effect nucleases (TALENs) and regularly clustered short palindromic repeat-associated protein (CRISPR-Cas9)) and review how these systems have allowed the generation of cellular and animal GKDs models and how they have contributed to shed light on many still unknown fields in GKDs. We also indicate the main obstacles limiting the application of these systems in a more efficient way. The information provided here will be useful to gain an accurate understanding of the technological advances in the field of genome editing for GKDs, as well as to serve as a guide for the selection of both the genome editing tool and the gene delivery method most suitable for the successful development of GKDs models. Full article
(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application Series 2)
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25 pages, 1384 KiB  
Review
Principles and Applications of CRISPR Toolkit in Virus Manipulation, Diagnosis, and Virus-Host Interactions
by Saleh Jamehdor, Sara Pajouhanfar, Sadaf Saba, Georges Uzan, Ali Teimoori and Sina Naserian
Cells 2022, 11(6), 999; https://0-doi-org.brum.beds.ac.uk/10.3390/cells11060999 - 15 Mar 2022
Cited by 3 | Viewed by 4756
Abstract
Viruses are one of the most important concerns for human health, and overcoming viral infections is a worldwide challenge. However, researchers have been trying to manipulate viral genomes to overcome various disorders, including cancer, for vaccine development purposes. CRISPR (clustered regularly interspaced short [...] Read more.
Viruses are one of the most important concerns for human health, and overcoming viral infections is a worldwide challenge. However, researchers have been trying to manipulate viral genomes to overcome various disorders, including cancer, for vaccine development purposes. CRISPR (clustered regularly interspaced short palindromic repeats) is becoming one of the most functional and widely used tools for RNA and DNA manipulation in multiple organisms. This approach has provided an unprecedented opportunity for creating simple, inexpensive, specific, targeted, accurate, and practical manipulations of viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus-1 (HIV-1), and vaccinia virus. Furthermore, this method can be used to make an effective and precise diagnosis of viral infections. Nevertheless, a valid and scientifically designed CRISPR system is critical to make more effective and accurate changes in viruses. In this review, we have focused on the best and the most effective ways to design sgRNA, gene knock-in(s), and gene knock-out(s) for virus-targeted manipulation. Furthermore, we have emphasized the application of CRISPR technology in virus diagnosis and in finding significant genes involved in virus-host interactions. Full article
(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application Series 2)
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24 pages, 1617 KiB  
Review
CRISPR-Cas and Its Wide-Ranging Applications: From Human Genome Editing to Environmental Implications, Technical Limitations, Hazards and Bioethical Issues
by Roberto Piergentili, Alessandro Del Rio, Fabrizio Signore, Federica Umani Ronchi, Enrico Marinelli and Simona Zaami
Cells 2021, 10(5), 969; https://0-doi-org.brum.beds.ac.uk/10.3390/cells10050969 - 21 Apr 2021
Cited by 14 | Viewed by 10382
Abstract
The CRISPR-Cas system is a powerful tool for in vivo editing the genome of most organisms, including man. During the years this technique has been applied in several fields, such as agriculture for crop upgrade and breeding including the creation of allergy-free foods, [...] Read more.
The CRISPR-Cas system is a powerful tool for in vivo editing the genome of most organisms, including man. During the years this technique has been applied in several fields, such as agriculture for crop upgrade and breeding including the creation of allergy-free foods, for eradicating pests, for the improvement of animal breeds, in the industry of bio-fuels and it can even be used as a basis for a cell-based recording apparatus. Possible applications in human health include the making of new medicines through the creation of genetically modified organisms, the treatment of viral infections, the control of pathogens, applications in clinical diagnostics and the cure of human genetic diseases, either caused by somatic (e.g., cancer) or inherited (mendelian disorders) mutations. One of the most divisive, possible uses of this system is the modification of human embryos, for the purpose of preventing or curing a human being before birth. However, the technology in this field is evolving faster than regulations and several concerns are raised by its enormous yet controversial potential. In this scenario, appropriate laws need to be issued and ethical guidelines must be developed, in order to properly assess advantages as well as risks of this approach. In this review, we summarize the potential of these genome editing techniques and their applications in human embryo treatment. We will analyze CRISPR-Cas limitations and the possible genome damage caused in the treated embryo. Finally, we will discuss how all this impacts the law, ethics and common sense. Full article
(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application Series 2)
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14 pages, 705 KiB  
Review
Modeling Non-Alcoholic Fatty Liver Disease (NAFLD) Using “Good-Fit” Genome-Editing Tools
by Uijin Kim, Nahyun Kim and Ha Youn Shin
Cells 2020, 9(12), 2572; https://0-doi-org.brum.beds.ac.uk/10.3390/cells9122572 - 01 Dec 2020
Cited by 4 | Viewed by 3425
Abstract
Non-alcoholic fatty liver disease (NAFLD), which affects both adults and children, is the most common liver disorder worldwide. NAFLD is characterized by excess fat accumulation in the liver in the absence of significant alcohol use. NAFLD is strongly associated with obesity, insulin resistance, [...] Read more.
Non-alcoholic fatty liver disease (NAFLD), which affects both adults and children, is the most common liver disorder worldwide. NAFLD is characterized by excess fat accumulation in the liver in the absence of significant alcohol use. NAFLD is strongly associated with obesity, insulin resistance, metabolic syndrome, as well as specific genetic polymorphisms. Severe NAFLD cases can further progress to cirrhosis, hepatocellular carcinoma (HCC), or cardiovascular complications. Here, we describe the pathophysiological features and critical genetic variants associated with NAFLD. Recent advances in genome-engineering technology have provided a new opportunity to generate in vitro and in vivo models that reflect the genetic abnormalities of NAFLD. We review the currently developed NAFLD models generated using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing. We further discuss unique features of CRISPR/Cas9 and Cas9 variants, including base editors and prime editor, that are useful for replicating genetic features specific to NAFLD. We also compare advantages and limitations of currently available methods for delivering genome-editing tools necessary for optimal genome editing. This review should provide helpful guidance for selecting “good fit” genome-editing tools and appropriate gene-delivery methods for the successful development of NAFLD models and clinical therapeutics. Full article
(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application Series 2)
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17 pages, 2406 KiB  
Review
CRISPR FokI Dead Cas9 System: Principles and Applications in Genome Engineering
by Maryam Saifaldeen, Dana E. Al-Ansari, Dindial Ramotar and Mustapha Aouida
Cells 2020, 9(11), 2518; https://0-doi-org.brum.beds.ac.uk/10.3390/cells9112518 - 21 Nov 2020
Cited by 20 | Viewed by 5116
Abstract
The identification of the robust clustered regularly interspersed short palindromic repeats (CRISPR) associated endonuclease (Cas9) system gene-editing tool has opened up a wide range of potential therapeutic applications that were restricted by more complex tools, including zinc finger nucleases (ZFNs) and transcription activator-like [...] Read more.
The identification of the robust clustered regularly interspersed short palindromic repeats (CRISPR) associated endonuclease (Cas9) system gene-editing tool has opened up a wide range of potential therapeutic applications that were restricted by more complex tools, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Nevertheless, the high frequency of CRISPR system off-target activity still limits its applications, and, thus, advanced strategies for highly specific CRISPR/Cas9-mediated genome editing are continuously under development including CRISPR–FokI dead Cas9 (fdCas9). fdCas9 system is derived from linking a FokI endonuclease catalytic domain to an inactive Cas9 protein and requires a pair of guide sgRNAs that bind to the sense and antisense strands of the DNA in a protospacer adjacent motif (PAM)-out orientation, with a defined spacer sequence range around the target site. The dimerization of FokI domains generates DNA double-strand breaks, which activates the DNA repair machinery and results in genomic edit. So far, all the engineered fdCas9 variants have shown promising gene-editing activities in human cells when compared to other platforms. Herein, we review the advantages of all published variants of fdCas9 and their current applications in genome engineering. Full article
(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application Series 2)
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