scholarly journals Highly efficient and safe genome editing by CRISPR-Cas12a using CRISPR RNA with a ribosyl-2′-O-methylated uridinylate-rich 3′-overhang in mouse zygotes

2020 ◽  
Vol 52 (11) ◽  
pp. 1823-1830
Author(s):  
Dae-In Ha ◽  
Jeong Mi Lee ◽  
Nan-Ee Lee ◽  
Daesik Kim ◽  
Jeong-Heon Ko ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Animal Models ◽  
Genome Editing ◽  
Effector Proteins ◽  
Eukaryotic Cells ◽  
Aav Vector ◽  
Chemically Modified ◽  
Highly Efficient ◽  
Mouse Zygotes ◽  
Vector Delivery

AbstractThe CRISPR-Cas12a system has been developed to harness highly specific genome editing in eukaryotic cells. Given the relatively small sizes of Cas12a genes, the system has been suggested to be most applicable to gene therapy using AAV vector delivery. Previously, we reported that a U-rich crRNA enabled highly efficient genome editing by the CRISPR-Cas12a system in eukaryotic cells. In this study, we introduced methoxyl modifications at C2 in riboses in the U-rich 3′-overhang of crRNA. When mixed with Cas12a effector proteins, the ribosyl-2′-O-methylated (2-OM) U-rich crRNA enabled improvement of dsDNA digestibility. Moreover, the chemically modified U-rich crRNA achieved very safe and highly specific genome editing in murine zygotes. The engineered CRISPR-Cas12a system is expected to facilitate the generation of various animal models. Moreover, the engineered crRNA was evaluated to further improve a CRISPR genome editing toolset.

scholarly journals Improving Precise CRISPR Genome Editing by Small Molecules: Is there a Magic Potion?

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1318 ◽  
Author(s):  
Nadja Bischoff ◽  
Sandra Wimberger ◽  
Marcello Maresca ◽  
Cord Brakebusch
Keyword(s):  
Gene Therapy ◽  
Dna Repair ◽  
Animal Models ◽  
Molecular Biology ◽  
Small Molecules ◽  
Genome Editing ◽  
Drug Targets ◽  
Targeted Mutagenesis ◽  
Dna Repair Mechanisms ◽  
Repair Mechanisms

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing has become a standard method in molecular biology, for the establishment of genetically modified cellular and animal models, for the identification and validation of drug targets in animals, and is heavily tested for use in gene therapy of humans. While the efficiency of CRISPR mediated gene targeting is much higher than of classical targeted mutagenesis, the efficiency of CRISPR genome editing to introduce defined changes into the genome is still low. Overcoming this problem will have a great impact on the use of CRISPR genome editing in academic and industrial research and the clinic. This review will present efforts to achieve this goal by small molecules, which modify the DNA repair mechanisms to facilitate the precise alteration of the genome.

scholarly journals Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules

2020 ◽  
Author(s):  
Arik Shams ◽  
Sean A. Higgins ◽  
Christof Fellmann ◽  
Thomas G. Laughlin ◽  
Benjamin L. Oakes ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Dna Binding ◽  
Genome Editing ◽  
Experimental Validation ◽  
Effector Proteins ◽  
Multidomain Proteins ◽  
General Technique ◽  
Accretion Process

AbstractProteins evolve through the modular rearrangement of elements known as domains. It is hypothesized that extant, multidomain proteins are the result of domain accretion, but there has been limited experimental validation of this idea. Here, we introduce a technique for genetic minimization by iterative size-exclusion and recombination (MISER) that comprehensively assays all possible deletions of a protein. Using MISER, we generated a deletion landscape for the CRISPR protein Cas9. We found that Cas9 can tolerate large single deletions to the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics.

scholarly journals Prime editing primarily induces undesired outcomes in mice

2020 ◽  
Author(s):  
Tomomi Aida ◽  
Jonathan J. Wilde ◽  
Lixin Yang ◽  
Yuanyuan Hou ◽  
Mengqi Li ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Frequency ◽  
Mouse Embryos ◽  
Biomedical Science ◽  
New Approach ◽  
Chemically Modified ◽  
Highly Efficient ◽  
Large Deletions

SummaryGenome editing has transformed biomedical science, but is still unpredictable and often induces undesired outcomes. Prime editing (PE) is a promising new approach due to its proposed flexibility and ability to avoid unwanted indels. Here, we show highly efficient PE-mediated genome editing in mammalian zygotes. Utilizing chemically modified guideRNAs, PE efficiently introduced 10 targeted modifications including substitutions, deletions, and insertions across 6 genes in mouse embryos. However, we unexpectedly observed a high frequency of undesired outcomes such as large deletions and found that these occurred more often than pure intended edits across all of the edits/genes. We show that undesired outcomes result from the double-nicking PE3 strategy, but that omission of the second nick largely ablates PE function. However, sequential double-nicking with PE3b, which is only applicable to a fraction of edits, eliminated undesired outcomes. Overall, our findings demonstrate the promising potential of PE for predictable, flexible, and highly efficient in vivo genome editing, but highlight the need for improved variations of PE before it is ready for widespread use.

scholarly journals Genome Editing Technologies: From Bench Side to Bedside

Acta Medica ◽  
2018 ◽  
Vol 49 (3) ◽  
pp. 30
Author(s):  
Fulya Yaylacıoğlu Tuncay ◽  
Pervin Rukiye Dinçer
Keyword(s):  
Gene Therapy ◽  
Animal Models ◽  
Genome Editing ◽  
Clinical Studies ◽  
Human Gene ◽  
Human Gene Therapy ◽  
Novel Technologies ◽  
Preclinical And Clinical Studies

The development of genome editing technologies has given the chance to researchers to manipulate any genomic sequences precisely. This ability is very useful for creating animal models to study human diseases in vivo; for easy creation of isogenic cell lines to study in vitro and most importantly for overcoming many disadvantages that the researchers faced during the human gene therapy trials. Here we review the basic mechanisms of genome editing technology and the four genome-editing platforms. We also discuss the applications of these novel technologies in preclinical and clinical studies in four groups according to the mechanism used, and lastly, summarize the problems in these technologies.

scholarly journals Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Hongyi Li ◽  
Yang Yang ◽  
Weiqi Hong ◽  
Mengyuan Huang ◽  
Min Wu ◽  
...  
Keyword(s):  
Animal Models ◽  
Genome Editing ◽  
Gene Editing ◽  
Basic Research ◽  
Human Diseases ◽  
Eukaryotic Cells ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Major Genome ◽  
Almost All

AbstractBased on engineered or bacterial nucleases, the development of genome editing technologies has opened up the possibility of directly targeting and modifying genomic sequences in almost all eukaryotic cells. Genome editing has extended our ability to elucidate the contribution of genetics to disease by promoting the creation of more accurate cellular and animal models of pathological processes and has begun to show extraordinary potential in a variety of fields, ranging from basic research to applied biotechnology and biomedical research. Recent progress in developing programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)–Cas-associated nucleases, has greatly expedited the progress of gene editing from concept to clinical practice. Here, we review recent advances of the three major genome editing technologies (ZFNs, TALENs, and CRISPR/Cas9) and discuss the applications of their derivative reagents as gene editing tools in various human diseases and potential future therapies, focusing on eukaryotic cells and animal models. Finally, we provide an overview of the clinical trials applying genome editing platforms for disease treatment and some of the challenges in the implementation of this technology.

scholarly journals Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Masakazu Hashimoto ◽  
Tatsuya Takemoto
Keyword(s):  
Genetic Analysis ◽  
Genome Editing ◽  
High Throughput ◽  
Large Scale ◽  
Mouse Embryos ◽  
Mutant Mice ◽  
Highly Efficient ◽  
Mouse Zygotes ◽  
Time Required

Abstract Recent use of the CRISPR/Cas9 system has dramatically reduced the time required to produce mutant mice, but the involvement of a time-consuming microinjection step still hampers its application for high-throughput genetic analysis. Here we developed a simple, highly efficient and large-scale genome editing method, in which the RNAs for the CRISPR/Cas9 system are electroporated into zygotes rather than microinjected. We used this method to perform single-stranded oligodeoxynucleotide (ssODN)-mediated knock-in in mouse embryos. This method facilitates large-scale genetic analysis in the mouse.

Genome-editing is promising for producing therapeutically relevant animal models for possible therapies for rare human diseases

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Gene Therapy ◽  
Animal Models ◽  
Genome Editing ◽  
Cultured Cells ◽  
Bone Marrow Cells ◽  
Somatic Cells ◽  
Long Term Effects ◽  
Clinical Gene Therapy ◽  
Regulatory Challenges

The CRISPR/Cas9 genome-editing method shows tremendous promise in developing therapeutically relevant animal models to evaluate potential treatments for uncommon human illnesses. While damaging mutations are commonly fixed in laboratories in cultured cells and patient-derived iPSCs, CRISPR/Cas9 genome editing has emerged as a feasible technique to target somatic cells in rare monogenic and other genetically defined human illnesses. Recent advances in CRISPR/Cas9 technology have also enabled clinical gene therapy investigations. To get FDA clearance, several commercially accessible CRISPR/Cas9-based products have undergone several rounds of clinical development. The worldwide gene editing industry is anticipated to increase quickly to suit the therapeutic demands of several unique diseases that lack viable therapies. While this approach has hastened the development of breakthrough rare disease medicines, several key problems remain unsolved regarding the effectiveness, safety and off-target consequences of CRISPR/Cas9 genome editing in patients. Furthermore, anticipated clinical uses of genome editing pose ethical and regulatory challenges. Most gene therapy applications target somatic cells, such as blood and bone marrow cells, so genome alterations are not inherited and passed onto future generations. Due to the unclear long-term effects of germline gene therapy, possible treatments targeting germ cells are deemed dangerous.

scholarly journals Progress in Gene Therapy to Prevent Retinal Ganglion Cell Loss in Glaucoma and Leber’s Hereditary Optic Neuropathy

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Sara E. Ratican ◽  
Andrew Osborne ◽  
Keith R. Martin
Keyword(s):  
Gene Therapy ◽  
Clinical Trials ◽  
Optic Nerve ◽  
Genome Editing ◽  
Optic Neuropathy ◽  
Single Gene ◽  
Leber’S Hereditary Optic Neuropathy ◽  
Leber's Hereditary Optic Neuropathy ◽  
Potential Use ◽  
Hereditary Optic Neuropathy

The eye is at the forefront of the application of gene therapy techniques to medicine. In the United States, a gene therapy treatment for Leber’s congenital amaurosis, a rare inherited retinal disease, recently became the first gene therapy to be approved by the FDA for the treatment of disease caused by mutations in a specific gene. Phase III clinical trials of gene therapy for other single-gene defect diseases of the retina and optic nerve are also currently underway. However, for optic nerve diseases not caused by single-gene defects, gene therapy strategies are likely to focus on slowing or preventing neuronal death through the expression of neuroprotective agents. In addition to these strategies, there has also been recent interest in the potential use of precise genome editing techniques to treat ocular disease. This review focuses on recent developments in gene therapy techniques for the treatment of glaucoma and Leber’s hereditary optic neuropathy (LHON). We discuss recent successes in clinical trials for the treatment of LHON using gene supplementation therapy, promising neuroprotective strategies that have been employed in animal models of glaucoma and the potential use of genome editing techniques in treating optic nerve disease.

scholarly journals An Efficient, Rapid, and Recyclable System for CRISPR-Mediated Genome Editing in Candida albicans

mSphere ◽  
2017 ◽  
Vol 2 (2) ◽  
Author(s):  
Namkha Nguyen ◽  
Morgan M. F. Quail ◽  
Aaron D. Hernday
Keyword(s):  
Candida Albicans ◽  
Genome Editing ◽  
Fungal Pathogen ◽  
Gene Knockout ◽  
Strain Engineering ◽  
Molecular Genetic Analysis ◽  
Content Type ◽  
Highly Efficient ◽  
Discovery Research ◽  
Gene Knockouts

ABSTRACT Candida albicans is the most common fungal pathogen of humans. Historically, molecular genetic analysis of this important pathogen has been hampered by the lack of stable plasmids or meiotic cell division, limited selectable markers, and inefficient methods for generating gene knockouts. The recent development of clustered regularly interspaced short palindromic repeat(s) (CRISPR)-based tools for use with C. albicans has opened the door to more efficient genome editing; however, previously reported systems have specific limitations. We report the development of an optimized CRISPR-based genome editing system for use with C. albicans. Our system is highly efficient, does not require molecular cloning, does not leave permanent markers in the genome, and supports rapid, precise genome editing in C. albicans. We also demonstrate the utility of our system for generating two independent homozygous gene knockouts in a single transformation and present a method for generating homozygous wild-type gene addbacks at the native locus. Furthermore, each step of our protocol is compatible with high-throughput strain engineering approaches, thus opening the door to the generation of a complete C. albicans gene knockout library. IMPORTANCE Candida albicans is the major fungal pathogen of humans and is the subject of intense biomedical and discovery research. Until recently, the pace of research in this field has been hampered by the lack of efficient methods for genome editing. We report the development of a highly efficient and flexible genome editing system for use with C. albicans. This system improves upon previously published C. albicans CRISPR systems and enables rapid, precise genome editing without the use of permanent markers. This new tool kit promises to expedite the pace of research on this important fungal pathogen.

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