CRISPR/Cas9 gene editing for genodermatoses: progress and perspectives

2019 ◽  
Vol 3 (3) ◽  
pp. 313-326 ◽  
Author(s):  
Gaetano Naso ◽  
Anastasia Petrova
Keyword(s):  
Gene Editing ◽  
Ex Vivo ◽  
Current Status ◽  
Transcription Activator ◽  
Base Editing ◽  
Novel Treatments ◽  
Ex Vivo Gene Therapy ◽  
Engineered Nucleases ◽  
Blistering Disease ◽  
Therapy Strategies

Abstract Genodermatoses constitute a clinically heterogeneous group of devastating genetic skin disorders. Currently, therapy options are largely limited to symptomatic treatments and although significant advances have been made in ex vivo gene therapy strategies, various limitations remain. However, the recent technical transformation of the genome editing field promises to overcome the hurdles associated with conventional gene addition approaches. In this review, we discuss the need for developing novel treatments and describe the current status of gene editing for genodermatoses, focusing on a severe blistering disease called epidermolysis bullosa (EB), for which significant progress has been made. Initial research utilized engineered nucleases such as transcription activator-like effector nucleases and meganucleases. However, over the last few years, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) have upstaged older generation gene editing tools. We examine different strategies for CRISPR/Cas9 application that can be employed depending on the type and position of the mutation as well as the mode of its inheritance. Promising developments in the field of base editing opens new avenues for precise correction of single base substitutions, common in EB and other genodermatoses. We also address the potential limitations and challenges such as safety concerns and delivery efficiency. This review gives an insight into the future of gene editing technologies for genodermatoses.

scholarly journals Gene editing and CRISPR in the clinic: current and future perspectives

2020 ◽  
Vol 40 (4) ◽  
Author(s):  
Matthew P. Hirakawa ◽  
Raga Krishnakumar ◽  
Jerilyn A. Timlin ◽  
James P. Carney ◽  
Kimberly S. Butler
Keyword(s):  
Clinical Trials ◽  
Genome Editing ◽  
Dna Sequences ◽  
Gene Editing ◽  
Ex Vivo ◽  
Scientific Basis ◽  
Current Status ◽  
Zinc Finger Nucleases ◽  
Transcription Activator

Abstract Genome editing technologies, particularly those based on zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspaced short palindromic repeat DNA sequences)/Cas9 are rapidly progressing into clinical trials. Most clinical use of CRISPR to date has focused on ex vivo gene editing of cells followed by their re-introduction back into the patient. The ex vivo editing approach is highly effective for many disease states, including cancers and sickle cell disease, but ideally genome editing would also be applied to diseases which require cell modification in vivo. However, in vivo use of CRISPR technologies can be confounded by problems such as off-target editing, inefficient or off-target delivery, and stimulation of counterproductive immune responses. Current research addressing these issues may provide new opportunities for use of CRISPR in the clinical space. In this review, we examine the current status and scientific basis of clinical trials featuring ZFNs, TALENs, and CRISPR-based genome editing, the known limitations of CRISPR use in humans, and the rapidly developing CRISPR engineering space that should lay the groundwork for further translation to clinical application.

scholarly journals Immunodeficient Rabbit Models: History, Current Status and Future Perspectives

2020 ◽  
Vol 10 (20) ◽  
pp. 7369
Author(s):  
Jun Song ◽  
Brooke Pallas ◽  
Dongshan Yang ◽  
Jifeng Zhang ◽  
Yash Agarwal ◽  
...  
Keyword(s):  
Gene Editing ◽  
Interleukin 2 ◽  
Current Status ◽  
Loss Of Function ◽  
Future Perspectives ◽  
Transcription Activator ◽  
Immune Genes ◽  
Forkhead Box ◽  
Recombination Activating Gene ◽  
Model Family

Production of immunodeficient (ID) models in non-murine animal species had been extremely challenging until the advent of gene-editing tools: first zinc finger nuclease (ZFN), then transcription activator-like effector nuclease (TALEN), and most recently clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR)/Cas9. We and others used those gene-editing tools to develop ID rabbits carrying a loss of function mutation in essential immune genes, such as forkhead box protein N1 (FOXN1), recombination activating gene 1/2 (RAG1/2), and interleukin 2 receptor subunit gamma (IL2RG). Like their mouse counterparts, ID rabbits have profound defects in their immune system and are prone to bacterial and pneumocystis infections without prophylactic antibiotics. In addition to their use as preclinical models for primary immunodeficient diseases, ID rabbits are expected to contribute significantly to regenerative medicine and cancer research, where they serve as recipients for allo- and xeno-grafts, with notable advantages over mouse models, including a longer lifespan and a much larger body size. Here we provide a concise review of the history and current status of the development of ID rabbits, as well as future perspectives of this new member in the animal model family.

Gene editing in tomatoes

2017 ◽  
Vol 1 (2) ◽  
pp. 183-191 ◽  
Author(s):  
Joyce Van Eck
Keyword(s):  
Gene Editing ◽  
Crop Improvement ◽  
Site Directed Mutagenesis ◽  
Dna Uptake ◽  
Food Crop ◽  
Transcription Activator ◽  
Base Editing ◽  
Crop Models ◽  
Functional Studies ◽  
Tomato Transformation

Tomato is an effective model plant species because it possesses the qualities necessary for genetic and functional studies, but is also a food crop making what is learned more translatable for crop improvement when compared with other non-food crop models. The availability of genome sequences for many genotypes and amenability to transformation methodologies (Agrobacterium-mediated, direct DNA uptake via protoplasts, biolistics) make tomato the perfect platform to study the application of gene-editing technologies. This review includes information related to tomato transformation methodology, one of the necessary requirements for gene editing, along with the status of site-directed mutagenesis by TALENs (transcription activator-like effector nucleases) and CRISPR/Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated Proteins). In addition to the reports on proof-of-concept experiments to demonstrate the feasibility of gene editing in tomato, there are many reports that show the power of these technologies for modification of traits, such as fruit characteristics (ripening, size, and parthenocarpy), pathogen susceptibility, architecture (plant and inflorescence), and metabolic engineering. Also highlighted in this review are reports on the application of a recent CRISPR technology called base editing that allows the modification of one base pair in a gene sequence and a strategy that takes advantage of a geminivirus replicon for delivery of DNA repair template.

scholarly journals Chloroplast and mitochondrial DNA editing in plants

Nature Plants ◽  
2021 ◽  
Author(s):  
Beum-Chang Kang ◽  
Su-Ji Bae ◽  
Seonghyun Lee ◽  
Jeong Sun Lee ◽  
Annie Kim ◽  
...  
Keyword(s):  
Gene Editing ◽  
Unmet Need ◽  
Rrna Gene ◽  
Transcription Activator ◽  
Base Editing ◽  
Dna Editing ◽  
Point Mutagenesis ◽  
Golden Gate Cloning ◽  
Expression Plasmids ◽  
Plant Organelles

AbstractPlant organelles including mitochondria and chloroplasts contain their own genomes, which encode many genes essential for respiration and photosynthesis, respectively. Gene editing in plant organelles, an unmet need for plant genetics and biotechnology, has been hampered by the lack of appropriate tools for targeting DNA in these organelles. In this study, we developed a Golden Gate cloning system1, composed of 16 expression plasmids (8 for the delivery of the resulting protein to mitochondria and the other 8 for delivery to chloroplasts) and 424 transcription activator-like effector subarray plasmids, to assemble DddA-derived cytosine base editor (DdCBE)2 plasmids and used the resulting DdCBEs to efficiently promote point mutagenesis in mitochondria and chloroplasts. Our DdCBEs induced base editing in lettuce or rapeseed calli at frequencies of up to 25% (mitochondria) and 38% (chloroplasts). We also showed DNA-free base editing in chloroplasts by delivering DdCBE mRNA to lettuce protoplasts to avoid off-target mutations caused by DdCBE-encoding plasmids. Furthermore, we generated lettuce calli and plantlets with edit frequencies of up to 99%, which were resistant to streptomycin or spectinomycin, by introducing a point mutation in the chloroplast 16S rRNA gene.

scholarly journals Gene editing for inflammatory disorders

2018 ◽  
Vol 78 (1) ◽  
pp. 6-15 ◽  
Author(s):  
David T Ewart ◽  
Erik J Peterson ◽  
Clifford J Steer
Keyword(s):  
T Cells ◽  
Genetic Modification ◽  
Gene Editing ◽  
Ex Vivo ◽  
Inflammatory Diseases ◽  
Target Cells ◽  
Base Editing ◽  
Monogenic Diseases ◽  
Autoimmune And Inflammatory Diseases ◽  
Induced Pluripotent

Technology for precise and efficient genetic editing is constantly evolving and is now capable of human clinical applications. Autoimmune and inflammatory diseases are chronic, disabling, sometimes life-threatening, conditions that feature heritable components. Both primary genetic lesions and the inflammatory pathobiology underlying these diseases represent fertile soil for new therapies based on the capabilities of gene editing. The ability to orchestrate precise targeted modifications to the genome will likely enable cell-based therapies for inflammatory diseases such as monogenic autoinflammatory disease, acquired autoimmune disease and for regenerative medicine in the setting of an inflammatory environment. Here, we discuss recent advances in genome editing and their evolving applications in immunoinflammatory diseases. Strengths and limitations of older genetic modification tools are compared with CRISPR/Cas9, base editing, RNA editing, targeted activators and repressors of transcription and targeted epigenetic modifiers. Commonly employed delivery vehicles to target cells or tissues of interest with genetic modification machinery, including viral, non-viral and cellular vectors, are described. Finally, applications in animal and human models of inflammatory diseases are discussed. Use of chimeric autoantigen receptor T cells, correction of monogenic diseases with genetically edited haematopoietic stem and progenitor cells, engineering of induced pluripotent stem cells and ex vivo expansion and modification of regulatory T cells for a range of chronic inflammatory diseases are reviewed.

scholarly journals Gene and Base Editing as a Therapeutic Option for Cystic Fibrosis—Learning from Other Diseases

Genes ◽  
2019 ◽  
Vol 10 (5) ◽  
pp. 387 ◽  
Author(s):  
Karen Mention ◽  
Lúcia Santos ◽  
Patrick T. Harrison
Keyword(s):  
Cystic Fibrosis ◽  
Clinical Trials ◽  
Animal Models ◽  
Gene Editing ◽  
Ex Vivo ◽  
Therapeutic Option ◽  
Autosomal Recessive Disorder ◽  
Cftr Gene ◽  
Base Editing

Cystic fibrosis (CF) is a monogenic autosomal recessive disorder caused by mutations in the CFTR gene. There are at least 346 disease-causing variants in the CFTR gene, but effective small-molecule therapies exist for only ~10% of them. One option to treat all mutations is CFTR cDNA-based therapy, but clinical trials to date have only been able to stabilise rather than improve lung function disease in patients. While cDNA-based therapy is already a clinical reality for a number of diseases, some animal studies have clearly established that precision genome editing can be significantly more effective than cDNA addition. These observations have led to a number of gene-editing clinical trials for a small number of such genetic disorders. To date, gene-editing strategies to correct CFTR mutations have been conducted exclusively in cell models, with no in vivo gene-editing studies yet described. Here, we highlight some of the key breakthroughs in in vivo and ex vivo gene and base editing in animal models for other diseases and discuss what might be learned from these studies in the development of editing strategies that may be applied to cystic fibrosis as a potential therapeutic approach. There are many hurdles that need to be overcome, including the in vivo delivery of editing machinery or successful engraftment of ex vivo-edited cells, as well as minimising potential off-target effects. However, a successful proof-of-concept study for gene or base editing in one or more of the available CF animal models could pave the way towards a long-term therapeutic strategy for this disease.

scholarly journals Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

Biology ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 530
Author(s):  
Marlo K. Thompson ◽  
Robert W. Sobol ◽  
Aishwarya Prakash
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Gene Editing ◽  
Adaptive Immune System ◽  
Zinc Finger Nucleases ◽  
Specific Gene ◽  
Target Sequence ◽  
Transcription Activator ◽  
Low Cytotoxicity

The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

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