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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biology".

Deadline for manuscript submissions: closed (31 August 2018) | Viewed by 126876

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Prof. Dr. Izuho Hatada

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Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan
Interests: genome editing; epigenome editing; epigenetics; genomics; microRNA; genomic imprinting; obesity
Special Issues, Collections and Topics in MDPI journals

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Dear Colleagues,

Conventional gene targeting methods using homologous recombination are generally used for making knockout mice. However, the efficiency of this technology is not high. Therefore, the method requires embryonic stem cells. A recent breakthrough is the development of site-specific endonucleases for selective genome cleavage. Because of this method’s high efficiency, these endonucleases make it possible to direct gene targeting using fertilized eggs. These enzymes include zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and Cas9 nucleases. The technology using these nucleases is called “genome editing.” By using these technologies, we can make knockout organisms, including for most animals and plants, which cannot be created via conventional methods. Through combination with other technologies, genome editing can generate many new technologies, such as epigenome editing, live imaging, and genome-wide screening. The field of genome editing is growing at a surprisingly rapid rate and has had a great impact on basic science, clinical applications, and breeding technologies. The main purpose of this Special Issue is to provide an open-source sharing of significant works in the field of genome editing, so as to archive new technological developments and applications.

Prof. Dr. Izuho Hatada
Guest Editor

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Keywords

genome editing
ZFN
TALEN
CRISPR/Cas
Cas9
crystallography
target design
knockout
knockin
homology-directed repair (HDR)
epigenome editing
live imaging
enChIP
genome-wide screening
pluripotent stem cells
iPS cells
gene therapy

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Genome Editing in International Journal of Molecular Sciences (16 articles)

Published Papers (14 papers)

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15 pages, 3905 KiB

Open AccessArticle

The Combinational Use of CRISPR/Cas9 and Targeted Toxin Technology Enables Efficient Isolation of Bi-Allelic Knockout Non-Human Mammalian Clones

by Satoshi Watanabe, Takayuki Sakurai, Shingo Nakamura, Kazuchika Miyoshi and Masahiro Sato

Int. J. Mol. Sci. 2018, 19(4), 1075; https://doi.org/10.3390/ijms19041075 - 04 Apr 2018

Cited by 9 | Viewed by 4859

Abstract

Recent advances in genome editing systems such as clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) have facilitated genomic modification in mammalian cells. However, most systems employ transient treatment with selective drugs such as puromycin to obtain the desired genome-edited cells, which often allows some untransfected cells to survive and decreases the efficiency of generating genome-edited cells. Here, we developed a novel targeted toxin-based drug-free selection system for the enrichment of genome-edited cells. Cells were transfected with three expression vectors, each of which carries a guide RNA (gRNA), humanized Cas9 (hCas9) gene, or Clostridium perfringens-derived endo-β-galactosidase C (EndoGalC) gene. Once EndoGalC is expressed in a cell, it digests the cell-surface α-Gal epitope, which is specifically recognized by BS-I-B₄ lectin (IB4). Three days after transfection, these cells were treated with cytotoxin saporin-conjugated IB4 (IB4SAP) for 30 min at 37 °C prior to cultivation in a normal medium. Untransfected cells and those weakly expressing EndoGalC will die due to the internalization of saporin. Cells transiently expressing EndoGalC strongly survive, and some of these surviving clones are expected to be genome-edited bi-allelic knockout (KO) clones due to their strong co-expression of gRNA and hCas9. When porcine α-1,3-galactosyltransferase gene, which can synthesize the α-Gal epitope, was attempted to be knocked out, 16.7% and 36.7% of the surviving clones were bi-allelic and mono-allelic knockout (KO) cells, respectively, which was in contrast to the isolation of clones in the absence of IB4SAP treatment. Namely, 0% and 13.3% of the resulting clones were bi-allelic and mono-allelic KO cells, respectively. A similar tendency was seen when other target genes such as DiGeorge syndrome critical region gene 2 and transforming growth factor-β receptor type 1 gene were targeted to be knocked out. Our results indicate that a combination of the CRISPR/Cas9 system and targeted toxin technology using IB4SAP allows efficient enrichment of genome-edited clones, particularly bi-allelic KO clones. Full article

(This article belongs to the Special Issue Genome Editing 2018)

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20 pages, 4633 KiB

Open AccessArticle

CRISPR/Cas9-Based Cellular Engineering for Targeted Gene Overexpression

by Mark J. Osborn, Christopher J. Lees, Amber N. McElroy, Sarah C. Merkel, Cindy R. Eide, Wendy Mathews, Colby J. Feser, Madison Tschann, Ron T. McElmury, Beau R. Webber, Chong Jai Kim, Bruce R. Blazar and Jakub Tolar

Int. J. Mol. Sci. 2018, 19(4), 946; https://doi.org/10.3390/ijms19040946 - 22 Mar 2018

Cited by 17 | Viewed by 9193

Abstract

Gene and cellular therapies hold tremendous promise as agents for treating genetic disorders. However, the effective delivery of genes, particularly large ones, and expression at therapeutic levels can be challenging in cells of clinical relevance. To address this engineering hurdle, we sought to employ the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to insert powerful regulatory elements upstream of an endogenous gene. We achieved robust activation of the COL7A1 gene in primary human umbilical cord blood CD34⁺ hematopoietic stem cells and peripheral blood T-cells. CD34⁺ cells retained their colony forming potential and, in a second engineering step, we disrupted the T-cell receptor complex in T-cells. These cellular populations are of high translational impact due to their engraftment potential, broad circulatory properties, and favorable immune profile that supports delivery to multiple recipients. This study demonstrates the feasibility of targeted knock in of a ubiquitous chromatin opening element, promoter, and marker gene that doubles as a suicide gene for precision gene activation. This system merges the specificity of gene editing with the high level, sustained gene expression achieved with gene therapy vectors. We predict that this design concept will be highly transferrable to most genes in multiple model systems representing a facile cellular engineering platform for promoting gene expression. Full article

(This article belongs to the Special Issue Genome Editing 2018)

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Open AccessArticle

Efficient Generation of Somatic Cell Nuclear Transfer-Competent Porcine Cells with Mutated Alleles at Multiple Target Loci by Using CRISPR/Cas9 Combined with Targeted Toxin-Based Selection System

by Masahiro Sato, Kazuchika Miyoshi, Shingo Nakamura, Masato Ohtsuka, Takayuki Sakurai, Satoshi Watanabe, Hiroaki Kawaguchi and Akihide Tanimoto

Int. J. Mol. Sci. 2017, 18(12), 2610; https://doi.org/10.3390/ijms18122610 - 04 Dec 2017

Cited by 5 | Viewed by 4602

Abstract

The recent advancement in genome editing such a CRISPR/Cas9 system has enabled isolation of cells with knocked multiple alleles through a one-step transfection. Somatic cell nuclear transfer (SCNT) has been frequently employed as one of the efficient tools for the production of genetically modified (GM) animals. To use GM cells as SCNT donor, efficient isolation of transfectants with mutations at multiple target loci is often required. The methods for the isolation of such GM cells largely rely on the use of drug selection-based approach using selectable genes; however, it is often difficult to isolate cells with mutations at multiple target loci. In this study, we used a novel approach for the efficient isolation of porcine cells with at least two target loci mutations by one-step introduction of CRISPR/Cas9-related components. A single guide (sg) RNA targeted to GGTA1 gene, involved in the synthesis of cell-surface α-Gal epitope (known as xenogenic antigen), is always a prerequisite. When the transfected cells were reacted with toxin-labeled BS-I-B₄ isolectin for 2 h at 37 °C to eliminate α-Gal epitope-expressing cells, the surviving clones lacked α-Gal epitope expression and were highly expected to exhibit induced mutations at another target loci. Analysis of these α-Gal epitope-negative surviving cells demonstrated a 100% occurrence of genome editing at target loci. SCNT using these cells as donors resulted in the production of cloned blastocysts with the genotype similar to that of the donor cells used. Thus, this novel system will be useful for SCNT-mediated acquisition of GM cloned piglets, in which multiple target loci may be mutated. Full article

(This article belongs to the Special Issue Genome Editing 2018)

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935 KiB

Open AccessArticle

Efficient Generation of Genome-Modified Mice Using Campylobacter jejuni-Derived CRISPR/Cas

by Wataru Fujii, Arisa Ikeda, Koji Sugiura and Kunihiko Naito

Int. J. Mol. Sci. 2017, 18(11), 2286; https://doi.org/10.3390/ijms18112286 - 31 Oct 2017

Cited by 5 | Viewed by 3541

Abstract

Mammalian zygote-mediated genome-engineering by Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas is currently used for the generation of genome-modified animals. Here, we report that a Campylobacter jejuni-derived orthologous CRISPR/Cas system recognizes a 5′-NNNVRYAC sequence as a protospacer-adjacent motif in mouse zygotes, and is applicable for efficient generation of knockout mice. Moreover, this novel CRISPR/Cas can be used for zygote-mediated knock-in at a unique locus, suggesting that this system could help to expand the feasibility of the zygote-mediated generation of genome-modified animals. Full article

(This article belongs to the Special Issue Genome Editing 2018)

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7903 KiB

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FGF-2b and h-PL Transform Duct and Non-Endocrine Human Pancreatic Cells into Endocrine Insulin Secreting Cells by Modulating Differentiating Genes

by Giulia Donadel, Donatella Pastore, David Della-Morte, Barbara Capuani, Marco F. Lombardo, Francesca Pacifici, Marco Bugliani, Fabio A. Grieco, Piero Marchetti and Davide Lauro

Int. J. Mol. Sci. 2017, 18(11), 2234; https://doi.org/10.3390/ijms18112234 - 25 Oct 2017

Cited by 10 | Viewed by 4303

Abstract

Background: Diabetes mellitus (DM) is a multifactorial disease orphan of a cure. Regenerative medicine has been proposed as novel strategy for DM therapy. Human fibroblast growth factor (FGF)-2b controls β-cell clusters via autocrine action, and human placental lactogen (hPL)-A increases functional β-cells. We hypothesized whether FGF-2b/hPL-A treatment induces β-cell differentiation from ductal/non-endocrine precursor(s) by modulating specific genes expression. Methods: Human pancreatic ductal-cells (PANC-1) and non-endocrine pancreatic cells were treated with FGF-2b plus hPL-A at 500 ng/mL. Cytofluorimetry and Immunofluorescence have been performed to detect expression of endocrine, ductal and acinar markers. Bromodeoxyuridine incorporation and annexin-V quantified cells proliferation and apoptosis. Insulin secretion was assessed by RIA kit, and electron microscopy analyzed islet-like clusters. Results: Increase in PANC-1 duct cells de-differentiation into islet-like aggregates was observed after FGF-2b/hPL-A treatment showing ultrastructure typical of islets-aggregates. These clusters, after stimulation with FGF-2b/hPL-A, had significant (p < 0.05) increase in insulin, C-peptide, pancreatic and duodenal homeobox 1 (PDX-1), Nkx2.2, Nkx6.1, somatostatin, glucagon, and glucose transporter 2 (Glut-2), compared with control cells. Markers of PANC-1 (Cytokeratin-19, MUC-1, CA19-9) were decreased (p < 0.05). These aggregates after treatment with FGF-2b/hPL-A significantly reduced levels of apoptosis. Conclusions: FGF-2b and hPL-A are promising candidates for regenerative therapy in DM by inducing de-differentiation of stem cells modulating pivotal endocrine genes. Full article

(This article belongs to the Special Issue Genome Editing 2018)

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2776 KiB

Open AccessArticle

Human Globozoospermia-Related Gene Spata16 Is Required for Sperm Formation Revealed by CRISPR/Cas9-Mediated Mouse Models

by Yoshitaka Fujihara, Asami Oji, Tamara Larasati, Kanako Kojima-Kita and Masahito Ikawa

Int. J. Mol. Sci. 2017, 18(10), 2208; https://doi.org/10.3390/ijms18102208 - 21 Oct 2017

Cited by 43 | Viewed by 7785

Abstract

A recent genetic analysis of infertile globozoospermic patients identified causative mutations in three genes: a protein interacting with C kinase 1 (PICK1), dpy 19-like 2 (DPY19L2), and spermatogenesis associated 16 (SPATA16). Although mouse models have clarified the physiological functions of Pick1 and Dpy19l2 during spermatogenesis, Spata16 remains to be determined. Globozoospermic patients carried a homozygous point mutation in SPATA16 at 848G→A/R283Q. We generated CRISPR/Cas9-mediated mutant mice with the same amino acid substitution in the fourth exon of Spata16 to analyze the mutation site at R284Q, which corresponded with R283Q of mutated human SPATA16. We found that the point mutation in Spata16 was not essential for male fertility; however, deletion of the fourth exon of Spata16 resulted in infertile male mice due to spermiogenic arrest but not globozoospermia. This study demonstrates that Spata16 is indispensable for male fertility in mice, as well as in humans, as revealed by CRISPR/Cas9-mediated mouse models. Full article

(This article belongs to the Special Issue Genome Editing 2018)

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CRISPR-Cas9 Mediated Telomere Removal Leads to Mitochondrial Stress and Protein Aggregation

by Hyojung Kim, Sangwoo Ham, Minkyung Jo, Gum Hwa Lee, Yun-Song Lee, Joo-Ho Shin and Yunjong Lee

Int. J. Mol. Sci. 2017, 18(10), 2093; https://doi.org/10.3390/ijms18102093 - 03 Oct 2017

Cited by 22 | Viewed by 8183

Abstract

Aging is considered the major risk factor for neurodegenerative diseases including Parkinson’s disease (PD). Telomere shortening is associated with cellular senescence. In this regard, pharmacological or genetic inhibition of telomerase activity has been used to model cellular aging. Here, we employed CRISPR-Cas9 technology to instantly remove the telomere to induce aging in a neuroblastoma cell line. Expression of both Cas9 and guide RNA targeting telomere repeats ablated the telomere, leading to retardation of cell proliferation. Instant deletion of telomere in SH-SY5Y cells impaired mitochondrial function with diminished mitochondrial respiration and cell viability. Supporting the pathological relevance of cell aging by CRISPR-Cas9 mediated telomere removal, alterations were observed in the levels of PD-associated proteins including PTEN-induced putative kinase 1, peroxisome proliferator-activated receptor γ coactivator 1-α, nuclear respiratory factor 1, parkin, and aminoacyl tRNA synthetase complex interacting multifunctional protein 2. Significantly, α-synuclein expression in the background of telomere removal led to the enhancement of protein aggregation, suggesting positive feed-forward interaction between aging and PD pathogenesis. Collectively, our results demonstrate that CRISPR-Cas9 can be used to efficiently model cellular aging and PD. Full article

(This article belongs to the Special Issue Genome Editing 2018)

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4760 KiB

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Arid1b Haploinsufficiency Causes Abnormal Brain Gene Expression and Autism-Related Behaviors in Mice

by Mihiro Shibutani, Takuro Horii, Hirotaka Shoji, Sumiyo Morita, Mika Kimura, Naomi Terawaki, Tsuyoshi Miyakawa and Izuho Hatada

Int. J. Mol. Sci. 2017, 18(9), 1872; https://doi.org/10.3390/ijms18091872 - 30 Aug 2017

Cited by 50 | Viewed by 8318

Abstract

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with core symptoms that include poor social communication, restricted interests, and repetitive behaviors. Several ASD mouse models exhibit impaired social interaction, anxiety-like behavior, and elevated perseveration. Large-scale whole exome sequencing studies identified many genes putatively associated with ASD. Like chromodomain helicase DNA binding protein 8 (CHD8), the most frequently mutated gene in individuals with ASD, the candidate gene AT-rich interaction domain 1B (ARID1B) encodes a chromatin remodeling factor. Arid1b heterozygous knockout (hKO) mice exhibited ASD-like traits related to social behavior, anxiety, and perseveration, in addition to associated features reported in some cases of ASD, such as reduced weight, impaired motor coordination, and hydrocephalus. Hydrocephalus was present in 5 of 91 hKO mice, while it was not observed in wild-type littermates (0 of 188). Genome-wide gene expression patterns in Arid1b hKO mice were similar to those in ASD patients and Chd8-haploinsufficient mice, an ASD model, and to developmental changes in gene expression in fast-spiking cells in the mouse brain. Our results suggest that Arid1b haploinsufficiency causes ASD-like phenotypes in mice. Full article

(This article belongs to the Special Issue Genome Editing 2018)

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2786 KiB

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CRISPR/Cas9-Mediated Correction of the FANCD1 Gene in Primary Patient Cells

by Karolina Skvarova Kramarzova, Mark J. Osborn, Beau R. Webber, Anthony P. DeFeo, Amber N. McElroy, Chong Jai Kim and Jakub Tolar

Int. J. Mol. Sci. 2017, 18(6), 1269; https://doi.org/10.3390/ijms18061269 - 14 Jun 2017

Cited by 26 | Viewed by 7392

Abstract

Fanconi anemia (FA) is an inherited condition characterized by impaired DNA repair, physical anomalies, bone marrow failure, and increased incidence of malignancy. Gene editing holds great potential to precisely correct the underlying genetic cause such that gene expression remains under the endogenous control mechanisms. This has been accomplished to date only in transformed cells or their reprogrammed induced pluripotent stem cell counterparts; however, it has not yet been reported in primary patient cells. Here we show the ability to correct a mutation in Fanconi anemia D1 (FANCD1) primary patient fibroblasts. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was employed to target and correct a FANCD1 gene deletion. Homologous recombination using an oligonucleotide donor was achieved and a pure population of modified cells was obtained by using inhibitors of poly adenosine diphosphate-ribose polymerase (poly ADP-ribose polymerase). FANCD1 function was restored and we did not observe any promiscuous cutting of the CRISPR/Cas9 at off target sites. This consideration is crucial in the context of the pre-malignant FA phenotype. Altogether we show the ability to correct a patient mutation in primary FANCD1 cells in a precise manner. These proof of principle studies support expanded application of gene editing for FA. Full article

(This article belongs to the Special Issue Genome Editing 2018)

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Review

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19 pages, 701 KiB

Open AccessReview

In Vivo Genome Editing as a Therapeutic Approach

by Beatrice Xuan Ho, Sharon Jia Hui Loh, Woon Khiong Chan and Boon Seng Soh

Int. J. Mol. Sci. 2018, 19(9), 2721; https://doi.org/10.3390/ijms19092721 - 12 Sep 2018

Cited by 58 | Viewed by 9095

Abstract

Genome editing has been well established as a genome engineering tool that enables researchers to establish causal linkages between genetic mutation and biological phenotypes, providing further understanding of the genetic manifestation of many debilitating diseases. More recently, the paradigm of genome editing technologies has evolved to include the correction of mutations that cause diseases via the use of nucleases such as zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and more recently, Cas9 nuclease. With the aim of reversing disease phenotypes, which arise from somatic gene mutations, current research focuses on the clinical translatability of correcting human genetic diseases in vivo, to provide long-term therapeutic benefits and potentially circumvent the limitations of in vivo cell replacement therapy. In this review, in addition to providing an overview of the various genome editing techniques available, we have also summarized several in vivo genome engineering strategies that have successfully demonstrated disease correction via in vivo genome editing. The various benefits and challenges faced in applying in vivo genome editing in humans will also be discussed. Full article

(This article belongs to the Special Issue Genome Editing 2018)

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19 pages, 32485 KiB

Open AccessReview

Applications of CRISPR/Cas System to Bacterial Metabolic Engineering

by Suhyung Cho, Jongoh Shin and Byung-Kwan Cho

Int. J. Mol. Sci. 2018, 19(4), 1089; https://doi.org/10.3390/ijms19041089 - 05 Apr 2018

Cited by 110 | Viewed by 36581

Abstract

The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) adaptive immune system has been extensively used for gene editing, including gene deletion, insertion, and replacement in bacterial and eukaryotic cells owing to its simple, rapid, and efficient activities in unprecedented resolution. Furthermore, the CRISPR interference (CRISPRi) system including deactivated Cas9 (dCas9) with inactivated endonuclease activity has been further investigated for regulation of the target gene transiently or constitutively, avoiding cell death by disruption of genome. This review discusses the applications of CRISPR/Cas for genome editing in various bacterial systems and their applications. In particular, CRISPR technology has been used for the production of metabolites of high industrial significance, including biochemical, biofuel, and pharmaceutical products/precursors in bacteria. Here, we focus on methods to increase the productivity and yield/titer scan by controlling metabolic flux through individual or combinatorial use of CRISPR/Cas and CRISPRi systems with introduction of synthetic pathway in industrially common bacteria including Escherichia coli. Further, we discuss additional useful applications of the CRISPR/Cas system, including its use in functional genomics. Full article

(This article belongs to the Special Issue Genome Editing 2018)

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14 pages, 1904 KiB

Open AccessReview

Application of CRISPR-Cas9 Based Genome-Wide Screening Approaches to Study Cellular Signalling Mechanisms

by Sumana Sharma and Evangelia Petsalaki

Int. J. Mol. Sci. 2018, 19(4), 933; https://doi.org/10.3390/ijms19040933 - 21 Mar 2018

Cited by 42 | Viewed by 9906

Abstract

The cellular signalling process is a highly complex mechanism, involving multiple players, which together orchestrate the cell’s response to environmental changes and perturbations. Given the multitude of genes that participate in the process of cellular signalling, its study in a genome-wide manner has proven challenging. Recent advances in gene editing technologies, including clustered regularly-interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) approaches, have opened new opportunities to investigate global regulatory signalling programs of cells in an unbiased manner. In this review, we focus on how the application of pooled genetic screening approaches using the CRISPR/Cas9 system has contributed to a systematic understanding of cellular signalling processes in normal and disease contexts. Full article

(This article belongs to the Special Issue Genome Editing 2018)

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565 KiB

Open AccessReview

Applications of Alternative Nucleases in the Age of CRISPR/Cas9

by Tuhin K. Guha and David R. Edgell

Int. J. Mol. Sci. 2017, 18(12), 2565; https://doi.org/10.3390/ijms18122565 - 29 Nov 2017

Cited by 24 | Viewed by 6849

Abstract

Breakthroughs in the development of programmable site-specific nucleases, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases (MNs), and most recently, the clustered regularly interspaced short palindromic repeats (CRISPR) associated proteins (including Cas9) have greatly enabled and accelerated genome editing. By targeting double-strand breaks to user-defined locations, the rates of DNA repair events are greatly enhanced relative to un-catalyzed events at the same sites. However, the underlying biology of each genome-editing nuclease influences the targeting potential, the spectrum of off-target cleavages, the ease-of-use, and the types of recombination events at targeted double-strand breaks. No single genome-editing nuclease is optimized for all possible applications. Here, we focus on the diversity of nuclease domains available for genome editing, highlighting biochemical properties and the potential applications that are best suited to each domain. Full article

(This article belongs to the Special Issue Genome Editing 2018)

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827 KiB

Open AccessReview

Genome Modification Technologies and Their Applications in Avian Species

by Hong Jo Lee, Young Min Kim, Tamao Ono and Jae Yong Han

Int. J. Mol. Sci. 2017, 18(11), 2245; https://doi.org/10.3390/ijms18112245 - 26 Oct 2017

Cited by 10 | Viewed by 5374

Abstract

The rapid development of genome modification technology has provided many great benefits in diverse areas of research and industry. Genome modification technologies have also been actively used in a variety of research areas and fields of industry in avian species. Transgenic technologies such as lentiviral systems and piggyBac transposition have been used to produce transgenic birds for diverse purposes. In recent years, newly developed programmable genome editing tools such as transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) have also been successfully adopted in avian systems with primordial germ cell (PGC)-mediated genome modification. These genome modification technologies are expected to be applied to practical uses beyond system development itself. The technologies could be used to enhance economic traits in poultry such as acquiring a disease resistance or producing functional proteins in eggs. Furthermore, novel avian models of human diseases or embryonic development could also be established for research purposes. In this review, we discuss diverse genome modification technologies used in avian species, and future applications of avian biotechnology. Full article

(This article belongs to the Special Issue Genome Editing 2018)

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