Applications of CRISPR/Cas Gene-Editing Technology in Fungi

Abstract Genome editing technology develop fast in recent years. The traditional gene-editing methods, including homologous recombination, zinc finger endonuclease, and transcription activator-like effector nuclease and so on, which have greatly promoted the research of genetics and molecular biology, have gradually showed their limitations such as low efficiency, high error rate, and complex design. In 2012,a new gene-editing technology, the CRISPR/Cas9 system, was setup based on the research of the immune responses to viruses from archaea and bacteria. Due to its advantages of high target efficiency, simple primer design, and wide application, CRISPR/Cas9 system, whose developers are awared the Nobel Prize in Chemistry this year, has become the dominant genomic editing technology in global academia and some pharmaceuticals. Here we briefly introduce the CRISPR/Cas system and its main applications in yeast, filamentous fungi and macrofungi, including single nucleotide, polygene and polyploid editing, yeast chromosome construction, yeast genome and yeast library construction, CRISPRa/CRISPRi-mediated, CRISPR platform of non-traditional yeast and regulation of metabolic pathway, to highlight the possible applications on fungal infection treatment and to promote the transformation and application of the CRISPR/Cas system in fungi.

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Concepts and tools for gene editing

Reproduction Fertility and Development ◽

10.1071/rd16396 ◽

2017 ◽

Vol 29 (1) ◽

pp. 1 ◽

Cited By ~ 6

Author(s):

Santiago Josa ◽

Davide Seruggia ◽

Almudena Fernández ◽

Lluis Montoliu

Keyword(s):

Dna Sequences ◽

Gene Editing ◽

Embryonic Stem ◽

Zinc Finger Nucleases ◽

Technological Advancement ◽

Transcription Activator ◽

Genetic Modifications ◽

Associated Proteins ◽

At Will ◽

Cas Gene

Gene editing is a relatively recent concept in the molecular biology field. Traditional genetic modifications in animals relied on a classical toolbox that, aside from some technical improvements and additions, remained unchanged for many years. Classical methods involved direct delivery of DNA sequences into embryos or the use of embryonic stem cells for those few species (mice and rats) where it was possible to establish them. For livestock, the advent of somatic cell nuclear transfer platforms provided alternative, but technically challenging, approaches for the genetic alteration of loci at will. However, the entire landscape changed with the appearance of different classes of genome editors, from initial zinc finger nucleases, to transcription activator-like effector nucleases and, most recently, with the development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas). Gene editing is currently achieved by CRISPR–Cas-mediated methods, and this technological advancement has boosted our capacity to generate almost any genetically altered animal that can be envisaged.

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Hayvan Islahında Güncel Bir Yaklaşım: CRISPR/Cas9 Genom Modifikasyon Sistemi

Turkish Journal of Agriculture - Food Science and Technology ◽

10.24925/turjaf.v4i12.1118-1122.873 ◽

2016 ◽

Vol 4 (12) ◽

pp. 1118

Author(s):

Fatih Bilgi ◽

Zeynep Demirtaş ◽

Levent Mercan

Keyword(s):

Immune System ◽

Genetic Structure ◽

Environmental Effects ◽

Gene Editing ◽

Zinc Finger Nucleases ◽

Transcription Activator ◽

Yield Performance ◽

Efficient Gene ◽

New Gene ◽

Genome Modifications

Genome modifications include potential about providing significant advantages on increasing yield performance and developing resistance to diseases. Gene editing methods that provides silencing or expressing of a gene which is an individual already has, have important potential for improving genetic structure without environmental effects. In recent times, new gene editing systems were developed. These are ZFNs (Zinc Finger Nucleases), TALENs (Transcription Activator-like Effector Nucleases) and CRISPR/Cas nuclease systems. CRISPR/Cas system is a microbial immune system that uses RNA guided nucleases for destroying genetic materials and its potential usage like a simple and efficient gene editing mechanism in animals is being evaluated recently. In this review, we summarized CRISPR/Cas9 system and its usability in animal breeding

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Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

Biology ◽

10.3390/biology10060530 ◽

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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Non-viral delivery of CRISPR–Cas9 complexes for targeted gene editing via a polymer delivery system

Gene Therapy ◽

10.1038/s41434-021-00282-6 ◽

2021 ◽

Author(s):

Jonathan O’Keeffe Ahern ◽

Irene Lara-Sáez ◽

Dezhong Zhou ◽

Rodolfo Murillas ◽

Jose Bonafont ◽

...

Keyword(s):

Delivery System ◽

Gene Editing ◽

Transfection Efficiency ◽

Attractive Alternative ◽

Safe Delivery ◽

Guide Rna ◽

Recessive Dystrophic Epidermolysis Bullosa ◽

Targeted Gene Editing ◽

Genomic Editing ◽

Polymeric Vectors

AbstractRecent advances in molecular biology have led to the CRISPR revolution, but the lack of an efficient and safe delivery system into cells and tissues continues to hinder clinical translation of CRISPR approaches. Polymeric vectors offer an attractive alternative to viruses as delivery vectors due to their large packaging capacity and safety profile. In this paper, we have demonstrated the potential use of a highly branched poly(β-amino ester) polymer, HPAE-EB, to enable genomic editing via CRISPRCas9-targeted genomic excision of exon 80 in the COL7A1 gene, through a dual-guide RNA sequence system. The biophysical properties of HPAE-EB were screened in a human embryonic 293 cell line (HEK293), to elucidate optimal conditions for efficient and cytocompatible delivery of a DNA construct encoding Cas9 along with two RNA guides, obtaining 15–20% target genomic excision. When translated to human recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes, transfection efficiency and targeted genomic excision dropped. However, upon delivery of CRISPR–Cas9 as a ribonucleoprotein complex, targeted genomic deletion of exon 80 was increased to over 40%. Our study provides renewed perspective for the further development of polymer delivery systems for application in the gene editing field in general, and specifically for the treatment of RDEB.

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Abstract 344: Finding the Achilles’ heel of Muscle Giant---TALEN-mediated Gene-editing in Zebrafish Titin

Circulation Research ◽

10.1161/res.117.suppl_1.344 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Yu-Huan Shih ◽

Xiaolei Xu

Keyword(s):

Alternative Splicing ◽

Gene Editing ◽

Muscle Development ◽

Exon Skipping ◽

Transcription Activator ◽

Detailed Characterization ◽

A Domains ◽

Underlying Mechanisms ◽

Sarcomere Assembly

Background: TITIN (TTN) has more than 300 exons and encodes a gigantic protein that is crucial for heart and muscle development. Mutations in TTN caused a variety of human diseases including cardiomyopathy and muscular dystrophy. Recently, dilated cardiomyopathy-associated mutations on TTN have been found more frequently in exons encoding A-band domains but less in exons encoding the N-terminal Z-disc domains, suggesting that mutations in different exons of TTN cause distinct consequences. To elucidate the underlying mechanisms, we leveraged the Transcription Activator-Like Effects Nuclease (TALEN) technology in zebrafish to introduce truncating mutations in different exons of ttn, and then study their effects on heart and somites. Results: We generated truncational mutations in different exons of zebrafish titins encoding Z-disc, N2B, Novex-3, and A domains, respectively. Because zebrafish contains two titin homologues, ttna and ttnb, we introduced mutations in both genes at the corresponding loci. While both Z-disc and A band mutations on ttna disrupted sarcomere assembly in heart and somites, Z-disc or A band mutations on ttnb only affect somites without affecting the heart. Interestingly, a Z-disc mutation on ttna resulted in milder phenotypes than an A-band mutation, while a Z-disc mutation on ttnb generated severer phenotypes than an A-band mutation. No phenotype was observed in the homozygous fish in either ttna-novex-3 or ttnb-N2B mutant fish. Conclusions: A spectrum of truncational mutations in ttna and ttnb has been generated in zebrafish using the TALEN technology. Mutations in different exons result in different phenotypes. Detailed characterization of these mutants and double mutants will be presented, which shall elicit distinct contribution of alternative splicing and exon skipping as two candidate mechanisms during pathogenesis of Titinopathies.

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An efficient and adaptable workflow for editing disease-relevant single nucleotide variants using CRISPR/Cas9

10.1101/2021.11.12.467071 ◽

2021 ◽

Author(s):

Inga Usher ◽

Lorena Ligammari ◽

Sara Ahrabi ◽

Emily Hepburn ◽

Calum Connolly ◽

...

Keyword(s):

Genetic Research ◽

Bone Cancer ◽

Illumina Miseq ◽

Genetic Alterations ◽

Single Nucleotide Variants ◽

Experimental Conditions ◽

Single Nucleotide ◽

Droplet Pcr ◽

Low Efficiency

Single nucleotide variants are the commonest genetic alterations in the human genome. At least 60,000 have been reported to be associated with disease. The CRISPR/Cas9 system has transformed genetic research, making it possible to edit single nucleotides and study the function of genetic variants in vitro. While significant advances have improved the efficiency of CRISPR/Cas9, the editing of single nucleotides remains challenging. There are two major obstacles: low efficiency of accurate editing and the isolation of these cells from a pool of cells with other editing outcomes. We present data from 85 transfections of induced pluripotent stem cells and an immortalised cell line, comparing the effects of altering CRISPR/Cas9 design and experimental conditions on rates of single nucleotide substitution. We targeted variants in TP53, which predispose to several cancers, and in TBXT which is implicated in the pathogenesis of the bone cancer, chordoma. We describe a scalable and adaptable workflow for single nucleotide editing that incorporates contemporary techniques including Illumina MiSeq sequencing, TaqMan qPCR and digital droplet PCR for screening transfected cells as well as quality control steps to mitigate against common pitfalls. This workflow can be applied to CRISPR/Cas9 and other genome editing systems to maximise experimental efficiency.

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Gene editing as a promising approach for respiratory diseases

Journal of Medical Genetics ◽

10.1136/jmedgenet-2017-104960 ◽

2018 ◽

Vol 55 (3) ◽

pp. 143-149 ◽

Cited By ~ 2

Author(s):

Yichun Bai ◽

Yang Liu ◽

Zhenlei Su ◽

Yana Ma ◽

Chonghua Ren ◽

...

Keyword(s):

Respiratory Diseases ◽

Gene Editing ◽

Gene Mutations ◽

Well Being ◽

Short Review ◽

Zinc Finger Nucleases ◽

Transcription Activator ◽

Mortality And Morbidity ◽

Chronic Respiratory Diseases ◽

Difficulty Breathing

Respiratory diseases, which are leading causes of mortality and morbidity in the world, are dysfunctions of the nasopharynx, the trachea, the bronchus, the lung and the pleural cavity. Symptoms of chronic respiratory diseases, such as cough, sneezing and difficulty breathing, may seriously affect the productivity, sleep quality and physical and mental well-being of patients, and patients with acute respiratory diseases may have difficulty breathing, anoxia and even life-threatening respiratory failure. Respiratory diseases are generally heterogeneous, with multifaceted causes including smoking, ageing, air pollution, infection and gene mutations. Clinically, a single pulmonary disease can exhibit more than one phenotype or coexist with multiple organ disorders. To correct abnormal function or repair injured respiratory tissues, one of the most promising techniques is to correct mutated genes by gene editing, as some gene mutations have been clearly demonstrated to be associated with genetic or heterogeneous respiratory diseases. Zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) systems are three innovative gene editing technologies developed recently. In this short review, we have summarised the structure and operating principles of the ZFNs, TALENs and CRISPR/Cas9 systems and their preclinical and clinical applications in respiratory diseases.

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CRISPR (See Genomic Editing; Gene Editing)

Dictionary of Global Bioethics ◽

10.1007/978-3-030-54161-3_180 ◽

2021 ◽

pp. 369-370

Author(s):

Henk ten Have ◽

Maria do Céu Patrão Neves

Keyword(s):

Gene Editing ◽

Genomic Editing

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Immunodeficient Rabbit Models: History, Current Status and Future Perspectives

Applied Sciences ◽

10.3390/app10207369 ◽

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.

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The Diversity of Genetic Outcomes from CRISPR/Cas Gene Editing is Regulated by the Length of the Symmetrical Donor DNA Template

Genes ◽

10.3390/genes11101160 ◽

2020 ◽

Vol 11 (10) ◽

pp. 1160

Author(s):

Amanda M. Hewes ◽

Brett M. Sansbury ◽

Eric B. Kmiec

Keyword(s):

Gene Editing ◽

Bacterial Cells ◽

Knock Out ◽

Viral Genomes ◽

Homology Directed Repair ◽

Established Cell ◽

Dna Template ◽

Donor Template ◽

Eukaryotic Genomes ◽

Cas Gene

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas gene editing systems have enabled molecular geneticists to manipulate prokaryotic and eukaryotic genomes with greater efficiency and precision. CRISPR/Cas provides adaptive immunity in bacterial cells by degrading invading viral genomes. By democratizing this activity into human cells, it is possible to knock out specific genes to disable their function and repair errors. The latter of these activities requires the participation of a single-stranded donor DNA template that provides the genetic information to execute correction in a process referred to as homology directed repair (HDR). Here, we utilized an established cell-free extract system to determine the influence that the donor DNA template length has on the diversity of products from CRISPR-directed gene editing. This model system enables us to view all outcomes of this reaction and reveals that donor template length can influence the efficiency of the reaction and the categories of error-prone products that accompany it. A careful measurement of the products revealed a category of error-prone events that contained the corrected template along with insertions and deletions (indels). Our data provides foundational information for those whose aim is to translate CRISPR/Cas from bench to bedside.

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