Therapeutic genome editing with engineered nucleases

SummaryTargeted genome editing with designer nucleases, such as zinc finger nucleases, TALE nucleases, and CRISPR-Cas nucleases, has heralded a new era in gene therapy. Genetic disorders, which have not been amenable to conventional gene-addition-type gene therapy approaches, such as disorders with dominant inheritance or diseases caused by mutations in tightly regulated genes, can now be treated by precise genome surgery. Moreover, engineered nucleases enable novel genetic interventions to fight infectious diseases or to improve cancer immunotherapies. Here, we review the development of the different classes of programmable nucleases, discuss the challenges and improvements in translating gene editing into clinical use, and give an outlook on what applications can expect to enter the clinic in the near future.

Download Full-text

Precision genome editing in the CRISPR era

Biochemistry and Cell Biology ◽

10.1139/bcb-2016-0137 ◽

2017 ◽

Vol 95 (2) ◽

pp. 187-201 ◽

Cited By ~ 49

Author(s):

Jayme Salsman ◽

Graham Dellaire

Keyword(s):

Gene Therapy ◽

Genome Editing ◽

Point Mutations ◽

Dna Double Strand Breaks ◽

End Joining ◽

Genetic Changes ◽

New Era ◽

Homology Directed Repair ◽

Repair Template ◽

Non Homologous End Joining

With the introduction of precision genome editing using CRISPR–Cas9 technology, we have entered a new era of genetic engineering and gene therapy. With RNA-guided endonucleases, such as Cas9, it is possible to engineer DNA double strand breaks (DSB) at specific genomic loci. DSB repair by the error-prone non-homologous end-joining (NHEJ) pathway can disrupt a target gene by generating insertions and deletions. Alternatively, Cas9-mediated DSBs can be repaired by homology-directed repair (HDR) using an homologous DNA repair template, thus allowing precise gene editing by incorporating genetic changes into the repair template. HDR can introduce gene sequences for protein epitope tags, delete genes, make point mutations, or alter enhancer and promoter activities. In anticipation of adapting this technology for gene therapy in human somatic cells, much focus has been placed on increasing the fidelity of CRISPR–Cas9 and increasing HDR efficiency to improve precision genome editing. In this review, we will discuss applications of CRISPR technology for gene inactivation and genome editing with a focus on approaches to enhancing CRISPR–Cas9-mediated HDR for the generation of cell and animal models, and conclude with a discussion of recent advances and challenges towards the application of this technology for gene therapy in humans.

Download Full-text

Application of CRISPR/Cas9 Genome Editing System in Cereal Crops

The Open Biotechnology Journal ◽

10.2174/1874070701913010173 ◽

2019 ◽

Vol 13 (1) ◽

pp. 173-179

Author(s):

V. Edwin Hillary ◽

S. Antony Ceasar

Keyword(s):

Genome Editing ◽

Genetic Research ◽

Cost Effective ◽

Zinc Finger Nucleases ◽

Cereal Crops ◽

Transcription Activator ◽

New Era ◽

Yield And Quality ◽

Recent Developments ◽

User Friendly

Recent developments in targeted genome editing accelerated genetic research and opened new potentials to improve the crops for better yields and quality. Genome editing techniques like Zinc Finger Nucleases (ZFN) and Transcription Activator-Like Effector Nucleases (TALENs) have been accustomed to target any gene of interest. However, these systems have some drawbacks as they are very expensive and time consuming with labor-intensive protein construction protocol. A new era of genome editing technology has a user-friendly tool which is termed as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein9 (Cas9), is an RNA based genome editing system involving a simple and cost-effective design of constructs. CRISPR/Cas9 system has been successfully applied in diverse crops for various genome editing approaches. In this review, we highlight the application of the CRISPR/Cas9 system in cereal crops including rice, wheat, maize, and sorghum to improve these crops for better yield and quality. Since cereal crops supply a major source of food to world populations, their improvement using recent genome editing tools like CRISPR/Cas9 is timely and crucial. The genome editing of cereal crops using the CRISPR/Cas9 system would help to overcome the adverse effects of agriculture and may aid in conserving food security in developing countries.

Download Full-text

CRISPR/Cas9-mediated genome editing has demonstrated significant promise for genetic correction in autologous hematopoietic stem/progenitor cells (HSPCs) and induced pluripotent stem cells (iPSCs)

10.31219/osf.io/xk54r ◽

2021 ◽

Author(s):

Moataz Dowaidar

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Induced Pluripotent Stem Cells ◽

Progenitor Cells ◽

Genome Editing ◽

Pluripotent Stem Cells ◽

Genetic Disorders ◽

Hematopoietic Stem ◽

Hematological Disorders ◽

Induced Pluripotent

According to current research, CRISPR/Cas9-mediated genome editing has shown enormous potential in the correction of genetic defects in autologous hematopoietic stem/progenitor cells (HSPCs) and induced pluripotent stem cells (iPSCs). Furthermore, the advancement of iPSC reprogramming technology as well as the CRISPR/Cas9 system has opened the door to new possibilities in the field of gene and cell therapy combinations. Despite the fact that there are a number of technological obstacles to overcome, CRISPR/Cas9 remains a promising therapeutic method with a great deal of potential for future gene therapy applications. Early success in treating hereditary hematological disorders opens the door to new options for treating other genetic disorders and constitutes a significant step forward in the development of gene therapy.

Download Full-text

Gene Therapy for Cystic Fibrosis: Progress and Challenges of Genome Editing

International Journal of Molecular Sciences ◽

10.3390/ijms21113903 ◽

2020 ◽

Vol 21 (11) ◽

pp. 3903 ◽

Cited By ~ 2

Author(s):

Giulia Maule ◽

Daniele Arosio ◽

Anna Cereseto

Keyword(s):

Cystic Fibrosis ◽

Gene Therapy ◽

Genome Editing ◽

Genetic Diseases ◽

Experimental Models ◽

Different Types ◽

Therapy Field ◽

Programmable Nucleases ◽

Cystic Fibrosis Transmembrane ◽

Genome Manipulation

Since the early days of its conceptualization and application, human gene transfer held the promise of a permanent solution to genetic diseases including cystic fibrosis (CF). This field went through alternated periods of enthusiasm and distrust. The development of refined technologies allowing site specific modification with programmable nucleases highly revived the gene therapy field. CRISPR nucleases and derived technologies tremendously facilitate genome manipulation offering diversified strategies to reverse mutations. Here we discuss the advancement of gene therapy, from therapeutic nucleic acids to genome editing techniques, designed to reverse genetic defects in CF. We provide a roadmap through technologies and strategies tailored to correct different types of mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, and their applications for the development of experimental models valuable for the advancement of CF therapies.

Download Full-text

CRISPR/Cas9 genome editing technology applications in biological and biomedical fields

10.31219/osf.io/ctqbe ◽

2021 ◽

Author(s):

Moataz Dowaidar

Keyword(s):

Gene Therapy ◽

Genome Editing ◽

Dna Sequences ◽

Recombinant Dna ◽

Genetic Disorders ◽

Specific Gene ◽

Recombinant Dna Technology ◽

P Gene ◽

A Genome ◽

Biomedical Fields

Gene therapy is a way of mending or replacing a gene in an undesirable or non-functional cell. Although used in both animals and plants, gene therapy is most usually linked with humans. Because there are so many genetic disorders caused by genetic abnormalities or unwanted gene expression, gene therapy is promising to treat and even cure many diseases. The scientific and pharmaceutical sectors are becoming interested in gene therapy.CRISPR was initially detected in prokaryotic organisms, bacteria and archaea genomes. Although nucleotide sequences are regularly discovered in many bacteria and archaea, the scientific community has not realized its importance for over a decade. People used these diverse DNA sequences as a diagnostic for genotyping and therefore considered them as a distinctive feature for each particular microbe. Scientists are beginning to comprehend that the CRISPR/Cas system is a prokaryotic defense system's adaptive immunity to viruses, due to the discovery of CRISPR-associated protein (Cas) and the use of recombinant DNA technology. This recently discovered CRISPR/Cas system was swiftly developed as a tool for editing a specific gene in a genome. Since 2012, CRISPR/Cas9 genome editing technology has been quickly researched and applied in several biological and biomedical fields. For various basic and practical research reasons, as well as biotechnological applications in agriculture and healthcare, CRISPR/Cas9 technology has altered and improved greatly over the past five years. Base editor invention and prime editing technology by fusing a Cas endonuclease with other functional enzymes, such as base converter enzymes, is one of several milestones in this fast progress.

Download Full-text

The clinical applications of genome editing in HIV

Blood ◽

10.1182/blood-2016-01-678144 ◽

2016 ◽

Vol 127 (21) ◽

pp. 2546-2552 ◽

Cited By ~ 34

Author(s):

Cathy X. Wang ◽

Paula M. Cannon

Keyword(s):

T Cells ◽

Genome Editing ◽

Viral Vectors ◽

Gene Cassette ◽

Clinical Findings ◽

Zinc Finger Nucleases ◽

Hematopoietic Stem ◽

Genetic Manipulations ◽

Hiv Therapy ◽

Engineered Nucleases

Abstract HIV/AIDS has long been at the forefront of the development of gene- and cell-based therapies. Although conventional gene therapy approaches typically involve the addition of anti-HIV genes to cells using semirandomly integrating viral vectors, newer genome editing technologies based on engineered nucleases are now allowing more precise genetic manipulations. The possible outcomes of genome editing include gene disruption, which has been most notably applied to the CCR5 coreceptor gene, or the introduction of small mutations or larger whole gene cassette insertions at a targeted locus. Disruption of CCR5 using zinc finger nucleases was the first-in-human application of genome editing and remains the most clinically advanced platform, with 7 completed or ongoing clinical trials in T cells and hematopoietic stem/progenitor cells (HSPCs). Here we review the laboratory and clinical findings of CCR5 editing in T cells and HSPCs for HIV therapy and summarize other promising genome editing approaches for future clinical development. In particular, recent advances in the delivery of genome editing reagents and the demonstration of highly efficient homology-directed editing in both T cells and HSPCs are expected to spur the development of even more sophisticated applications of this technology for HIV therapy.

Download Full-text

Myoediting: Toward Prevention of Muscular Dystrophy by Therapeutic Genome Editing

Physiological Reviews ◽

10.1152/physrev.00046.2017 ◽

2018 ◽

Vol 98 (3) ◽

pp. 1205-1240 ◽

Cited By ~ 13

Author(s):

Yu Zhang ◽

Chengzu Long ◽

Rhonda Bassel-Duby ◽

Eric N. Olson

Keyword(s):

Genome Editing ◽

Genome Engineering ◽

Genetic Disorders ◽

Premature Death ◽

Muscular Dystrophies ◽

Genetic Mutations ◽

Secondary Effects ◽

Disease Treatment ◽

Programmable Nucleases

Muscular dystrophies represent a large group of genetic disorders that significantly impair quality of life and often progress to premature death. There is no effective treatment for these debilitating diseases. Most therapies, developed to date, focus on alleviating the symptoms or targeting the secondary effects, while the underlying gene mutation is still present in the human genome. The discovery and application of programmable nucleases for site-specific DNA double-stranded breaks provides a powerful tool for precise genome engineering. In particular, the CRISPR/Cas system has revolutionized the genome editing field and is providing a new path for disease treatment by targeting the disease-causing genetic mutations. In this review, we provide a historical overview of genome-editing technologies, summarize the most recent advances, and discuss potential strategies and challenges for permanently correcting genetic mutations that cause muscular dystrophies.

Download Full-text

Comparison of the Feasibility, Efficiency, and Safety of Genome Editing Technologies

International Journal of Molecular Sciences ◽

10.3390/ijms221910355 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10355

Author(s):

Nicolás González González Castro ◽

Jan Bjelic ◽

Gunya Malhotra ◽

Cong Huang ◽

Salman Hasan Alsaffar

Keyword(s):

Genome Editing ◽

Zinc Finger Nucleases ◽

Clinical Settings ◽

Transcription Activator ◽

Entire Genome ◽

Rational Decision Making ◽

Programmable Nucleases ◽

High Degree ◽

Detection Strategies ◽

Selection Of

Recent advances in programmable nucleases including meganucleases (MNs), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) have propelled genome editing from explorative research to clinical and industrial settings. Each technology, however, features distinct modes of action that unevenly impact their applicability across the entire genome and are often tested under significantly different conditions. While CRISPR-Cas is currently leading the field due to its versatility, quick adoption, and high degree of support, it is not without limitations. Currently, no technology can be regarded as ideal or even applicable to every case as the context dictates the best approach for genetic modification within a target organism. In this review, we implement a four-pillar framework (context, feasibility, efficiency, and safety) to assess the main genome editing platforms, as a basis for rational decision-making by an expanding base of users, regulators, and consumers. Beyond carefully considering their specific use case with the assessment framework proposed here, we urge stakeholders interested in genome editing to independently validate the parameters of their chosen platform prior to commitment. Furthermore, safety across all applications, particularly in clinical settings, is a paramount consideration and comprehensive off-target detection strategies should be incorporated within workflows to address this. Often neglected aspects such as immunogenicity and the inadvertent selection of mutants deficient for DNA repair pathways must also be considered.

Download Full-text