Evaluating and Enhancing Target Specificity of Gene-Editing Nucleases and Deaminases

2019 ◽  
Vol 88 (1) ◽  
pp. 191-220 ◽  
Author(s):  
Daesik Kim ◽  
Kevin Luk ◽  
Scot A. Wolfe ◽  
Jin-Soo Kim
Keyword(s):  
Dna Sequences ◽  
Gene Editing ◽  
Genomic Rearrangements ◽  
Zinc Finger Nucleases ◽  
Collateral Damage ◽  
Transcription Activator ◽  
Genome Wide ◽  
Target Sites ◽  
Programmable Nucleases ◽  
Target Activity

Programmable nucleases and deaminases, which include zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR RNA-guided nucleases, and RNA-guided base editors, are now widely employed for the targeted modification of genomes in cells and organisms. These gene-editing tools hold tremendous promise for therapeutic applications. Importantly, these nucleases and deaminases may display off-target activity through the recognition of near-cognate DNA sequences to their target sites, resulting in collateral damage to the genome in the form of local mutagenesis or genomic rearrangements. For therapeutic genome-editing applications with these classes of programmable enzymes, it is essential to measure and limit genome-wide off-target activity. Herein, we discuss the key determinants of off-target activity for these systems. We describe various cell-based and cell-free methods for identifying genome-wide off-target sites and diverse strategies that have been developed for reducing the off-target activity of programmable gene-editing enzymes.

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.

Concepts and tools for gene editing

2017 ◽  
Vol 29 (1) ◽  
pp. 1 ◽  
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.

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.

Gene editing as a promising approach for respiratory diseases

2018 ◽  
Vol 55 (3) ◽  
pp. 143-149 ◽  
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.

scholarly journals The Role of Gene Editing in Neurodegenerative Diseases

2018 ◽  
Vol 27 (3) ◽  
pp. 364-378 ◽  
Author(s):  
Hueng-Chuen Fan ◽  
Ching-Shiang Chi ◽  
Yih-Jing Lee ◽  
Jeng-Dau Tsai ◽  
Shinn-Zong Lin ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Gene Editing ◽  
Clinical Manifestations ◽  
Zinc Finger Nucleases ◽  
Diagnostic Tools ◽  
Pathological Feature ◽  
Transcription Activator ◽  
Substantial Impact ◽  
Parkinson’S Diseases ◽  
The Impact

Neurodegenerative diseases (NDs), at least including Alzheimer’s, Huntington’s, and Parkinson’s diseases, have become the most dreaded maladies because there are no precise diagnostic tools or definite treatments for these debilitating diseases. The increased prevalence and a substantial impact on the social–economic and medical care of NDs propel governments to develop policies to counteract the impact. Although the etiologies of NDs are still unknown, growing evidence suggests that genetic, cellular, and circuit alternations may cause the generation of abnormal misfolded proteins, which uncontrolledly accumulate to damage and eventually overwhelm the protein-disposal mechanisms of these neurons, leading to a common pathological feature of NDs. If the functions and the connectivity can be restored, alterations and accumulated damages may improve. The gene-editing tools including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats–associated nucleases (CRISPR/CAS) have emerged as a novel tool not only for generating specific ND animal models for interrogating the mechanisms and screening potential drugs against NDs but also for the editing sequence-specific genes to help patients with NDs to regain function and connectivity. This review introduces the clinical manifestations of three distinct NDs and the applications of the gene-editing technology on these debilitating diseases.

scholarly journals Genome editing for blood disorders: state of the art and recent advances

2019 ◽  
Vol 3 (3) ◽  
pp. 289-299 ◽  
Author(s):  
Marianna Romito ◽  
Rajeev Rai ◽  
Adrian J. Thrasher ◽  
Alessia Cavazza
Keyword(s):  
Gene Editing ◽  
Therapeutic Potential ◽  
State Of The Art ◽  
Clinical Success ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Blood Disorders ◽  
Flexible Tool ◽  
Wide Range ◽  
Made In

Abstract In recent years, tremendous advances have been made in the use of gene editing to precisely engineer the genome. This technology relies on the activity of a wide range of nuclease platforms — such as zinc-finger nucleases, transcription activator-like effector nucleases, and the CRISPR–Cas system — that can cleave and repair specific DNA regions, providing a unique and flexible tool to study gene function and correct disease-causing mutations. Preclinical studies using gene editing to tackle genetic and infectious diseases have highlighted the therapeutic potential of this technology. This review summarizes the progresses made towards the development of gene editing tools for the treatment of haematological disorders and the hurdles that need to be overcome to achieve clinical success.

scholarly journals Genomic DNA transposition induced by human PGBD5

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Anton G Henssen ◽  
Elizabeth Henaff ◽  
Eileen Jiang ◽  
Amy R Eisenberg ◽  
Julianne R Carson ◽  
...  
Keyword(s):  
Transposable Element ◽  
Dna Sequences ◽  
Genomic Dna ◽  
Biological Function ◽  
Human Cells ◽  
Genomic Rearrangements ◽  
Dna Transposition ◽  
Genome Wide ◽  
Human Genes ◽  
Evolutionarily Conserved

Transposons are mobile genetic elements that are found in nearly all organisms, including humans. Mobilization of DNA transposons by transposase enzymes can cause genomic rearrangements, but our knowledge of human genes derived from transposases is limited. In this study, we find that the protein encoded by human PGBD5, the most evolutionarily conserved transposable element-derived gene in vertebrates, can induce stereotypical cut-and-paste DNA transposition in human cells. Genomic integration activity of PGBD5 requires distinct aspartic acid residues in its transposase domain, and specific DNA sequences containing inverted terminal repeats with similarity to piggyBac transposons. DNA transposition catalyzed by PGBD5 in human cells occurs genome-wide, with precise transposon excision and preference for insertion at TTAA sites. The apparent conservation of DNA transposition activity by PGBD5 suggests that genomic remodeling contributes to its biological function.

scholarly journals CRISPR-Cas Technology: Emerging Applications in Clinical Microbiology and Infectious Diseases

2021 ◽  
Vol 14 (11) ◽  
pp. 1171
Author(s):  
Sahar Serajian ◽  
Ehsan Ahmadpour ◽  
Sonia M. Rodrigues Oliveira ◽  
Maria de Lourdes Pereira ◽  
Siamak Heidarzadeh
Keyword(s):  
Infectious Diseases ◽  
Clinical Microbiology ◽  
Gene Editing ◽  
Zinc Finger Nucleases ◽  
Homing Endonucleases ◽  
Transcription Activator ◽  
Practical Applications ◽  
Novel Technologies ◽  
Resistant Strains ◽  
Drug Resistant Strains

Through the years, many promising tools for gene editing have been developed including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR-associated protein 9 (Cas9), and homing endonucleases (HEs). These novel technologies are now leading new scientific advancements and practical applications at an inimitable speed. While most work has been performed in eukaryotes, CRISPR systems also enable tools to understand and engineer bacteria. The increase in the number of multi-drug resistant strains highlights a necessity for more innovative approaches to the diagnosis and treatment of infections. CRISPR has given scientists a glimmer of hope in this area that can provide a novel tool to fight against antimicrobial resistance. This system can provide useful information about the functions of genes and aid us to find potential targets for antimicrobials. This paper discusses the emerging use of CRISPR-Cas systems in the fields of clinical microbiology and infectious diseases with a particular emphasis on future prospects.

scholarly journals Bovine Genome Analysis to Unravel the Location and Feature of Target Sites of RNA-Guided Hyperactivated Recombinase Gin with Spacer Length Six

2022 ◽  
Author(s):  
Shalu Kumari Pathak ◽  
Arvind Sonwane ◽  
Subodh Kumar
Keyword(s):  
Dna Sequences ◽  
Genomic Sequence ◽  
Sequence Data ◽  
Bovine Genome ◽  
Search Pattern ◽  
Spacer Length ◽  
Guide Rna ◽  
Target Sites ◽  
Emboss Package ◽  
Programmable Nucleases

Background: Programmable nucleases are very promising tools of genome editing (GE), but they suffer from limitations including potential risk of genotoxicity which led to the exploration of safer approach of GE based on RNA-guided recombinase (RGR) platform. RNA-guided recombinase (RGR) platform operates on a typical recognition or target site comprised of the minimal pseudo-core recombinase site, a 5 to 6-base pair spacer flanking it and whole this central region is flanked by two guide RNA-specified DNA sequences or Cas9 binding sites followed by protospacer adjacent motifs (PAMs). Methods: The current study focuses on analysis of entire cattle genome to prepare a detailed map of target sites for RNA-guided hyperactivated recombinase Gin with spacer length six. For this, chromosome wise whole genomic sequence data was retrieved from Ensembl. After that search pattern for recombinase Gin with spacer length six was designed. By using this search pattern, RGR target sites were located by using dreg program of Emboss package. Result: Total number of RGR target sites identified in bovine genome for recombinase Gin was 677 with spacer length six. It was also investigated that whether these RGR target sites are present with in any gene or not and it was found that RGR target sites lies in both genic and intergenic region. Besides this, description of genes in context with these target sites was identified.

scholarly journals Genomic DNA transposition induced by human PGBD5

2015 ◽  
Author(s):  
Anton Henssen ◽  
Elizabeth Henaff ◽  
Eileen Jiang ◽  
Amy R Eisenberg ◽  
Julianne R Carson ◽  
...  
Keyword(s):  
Transposable Element ◽  
Dna Sequences ◽  
Genomic Dna ◽  
Biological Function ◽  
Human Cells ◽  
Genomic Rearrangements ◽  
Dna Transposition ◽  
Genome Wide ◽  
Human Genes ◽  
Evolutionarily Conserved

Transposons are mobile genetic elements that are found in nearly all organisms, including humans. Mobilization of DNA transposons by transposase enzymes can cause genomic rearrangements, but our knowledge of human genes derived from transposases is limited. Here, we find that the protein encoded by human PGBD5, the most evolutionarily conserved transposable element-derived gene in chordates, can induce stereotypical cut-and-paste DNA transposition in human cells. Genomic integration activity of PGBD5 requires distinct aspartic acid residues in its transposase domain, and specific DNA sequences with inverted terminal repeats with similarity to piggyBac transposons. DNA transposition catalyzed by PGBD5 in human cells occurs genome-wide, with precise transposon excision and preference for insertion at TTAA sites. The apparent conservation of DNA transposition activity by PGBD5 raises the possibility that genomic remodeling may contribute to its biological function.

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