A therapeutic genome editing primer for cardiologist

Genome editing, or genome engineering is a type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of a living organism using engineered nucleases, or molecular scissors. Genome editing is being rapidly adopted into all fields of biomedical research, including the cardiovascular field, where it has facilitated a greater understanding of lipid metabolism, electrophysiology, cardiomyopathies, and other cardiovascular disorders, has helped to create a wider variety of cellular and animal models, and has opened the door to a new class of therapies. In this review, we discuss the applications of in vivo genome-editing therapies for cardiovascular disorder.

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Dynamic Genome Editing Using In Vivo Synthesized Donor ssDNA in Escherichia coli

Cells ◽

10.3390/cells9020467 ◽

2020 ◽

Vol 9 (2) ◽

pp. 467

Author(s):

Min Hao ◽

Zhaoguan Wang ◽

Hongyan Qiao ◽

Peng Yin ◽

Jianjun Qiao ◽

...

Keyword(s):

Escherichia Coli ◽

Genome Editing ◽

Genome Engineering ◽

Single Gene ◽

Rolling Circle Replication ◽

Rolling Circle ◽

Cas9 Nuclease ◽

Dynamic Genome ◽

Circular Ssdna

As a key element of genome editing, donor DNA introduces the desired exogenous sequence while working with other crucial machinery such as CRISPR-Cas or recombinases. However, current methods for the delivery of donor DNA into cells are both inefficient and complicated. Here, we developed a new methodology that utilizes rolling circle replication and Cas9 mediated (RC-Cas-mediated) in vivo single strand DNA (ssDNA) synthesis. A single-gene rolling circle DNA replication system from Gram-negative bacteria was engineered to produce circular ssDNA from a Gram-positive parent plasmid at a designed sequence in Escherichia coli. Furthermore, it was demonstrated that the desired linear ssDNA fragment could be cut out using CRISPR-associated protein 9 (CRISPR-Cas9) nuclease and combined with lambda Red recombinase as donor for precise genome engineering. Various donor ssDNA fragments from hundreds to thousands of nucleotides in length were synthesized in E. coli cells, allowing successive genome editing in growing cells. We hope that this RC-Cas-mediated in vivo ssDNA on-site synthesis system will be widely adopted as a useful new tool for dynamic genome editing.

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Advances in therapeutic application of CRISPR-Cas9

Briefings in Functional Genomics ◽

10.1093/bfgp/elz031 ◽

2019 ◽

Vol 19 (3) ◽

pp. 164-174 ◽

Cited By ~ 3

Author(s):

Jinyu Sun ◽

Jianchu Wang ◽

Donghui Zheng ◽

Xiaorong Hu

Keyword(s):

Gene Therapy ◽

Genome Editing ◽

Malignant Tumor ◽

Gene Editing ◽

Genome Engineering ◽

Therapeutic Application ◽

Adaptive Immune ◽

Efficient Gene

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is one of the most versatile and efficient gene editing technologies, which is derived from adaptive immune strategies for bacteria and archaea. With the remarkable development of programmable nuclease-based genome engineering these years, CRISPR-Cas9 system has developed quickly in recent 5 years and has been widely applied in countless areas, including genome editing, gene function investigation and gene therapy both in vitro and in vivo. In this paper, we briefly introduce the mechanisms of CRISPR-Cas9 tool in genome editing. More importantly, we review the recent therapeutic application of CRISPR-Cas9 in various diseases, including hematologic diseases, infectious diseases and malignant tumor. Finally, we discuss the current challenges and consider thoughtfully what advances are required in order to further develop the therapeutic application of CRISPR-Cas9 in the future.

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All-in-One Adeno-associated Virus Delivery and Genome Editing by Neisseria meningitidis Cas9 in vivo

10.1101/295055 ◽

2018 ◽

Cited By ~ 1

Author(s):

Raed Ibraheim ◽

Chun-Qing Song ◽

Aamir Mir ◽

Nadia Amrani ◽

Wen Xue ◽

...

Keyword(s):

Gene Therapy ◽

Neisseria Meningitidis ◽

Genome Editing ◽

Viral Vectors ◽

Genome Engineering ◽

Degradation Pathway ◽

Type I ◽

Guide Rna ◽

Hereditary Tyrosinemia

AbstractClustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) have recently opened a new avenue for gene therapy. Cas9 nuclease guided by a single-guide RNA (sgRNA) has been extensively used for genome editing. Currently, three Cas9 orthologs have been adapted for in vivo genome engineering applications: SpyCas9, SauCas9 and CjeCas9. However, additional in vivo editing platforms are needed, in part to enable a greater range of sequences to be accessed via viral vectors, especially those in which Cas9 and sgRNA are combined into a single vector genome. Here, we present an additional in vivo editing platform using Neisseria meningitidis Cas9 (NmeCas9). NmeCas9 is compact, edits with high accuracy, and possesses a distinct PAM, making it an excellent candidate for safe gene therapy applications. We find that NmeCas9 can be used to target the Pcsk9 and Hpd genes in mice. Using tail vein hydrodynamic-based delivery of NmeCas9 plasmid to target the Hpd gene, we successfully reprogrammed the tyrosine degradation pathway in Hereditary Tyrosinemia Type I mice. More importantly, we delivered NmeCas9 with its single-guide RNA in a single recombinant adeno-associated vector (rAAV) to target Pcsk9, resulting in lower cholesterol levels in mice. This all-in-one vector yielded >35% gene modification after two weeks of vector administration, with minimal off-target cleavage in vivo. Our findings indicate that NmeCas9 can facilitate future efforts to correct disease-causing mutations by expanding the targeting scope of RNA-guided nucleases.

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Genome editing in plants using CRISPR type I-D nuclease

Communications Biology ◽

10.1038/s42003-020-01366-6 ◽

2020 ◽

Vol 3 (1) ◽

Author(s):

Keishi Osakabe ◽

Naoki Wada ◽

Tomoko Miyaji ◽

Emi Murakami ◽

Kazuya Marui ◽

...

Keyword(s):

Genome Editing ◽

First Generation ◽

Genome Engineering ◽

Targeted Mutagenesis ◽

Plant Genome ◽

Type I ◽

Crispr System ◽

Target Dna ◽

Short Indels

Abstract Genome editing in plants has advanced greatly by applying the clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas system, especially CRISPR-Cas9. However, CRISPR type I—the most abundant CRISPR system in bacteria—has not been exploited for plant genome modification. In type I CRISPR-Cas systems, e.g., type I-E, Cas3 nucleases degrade the target DNA in mammals. Here, we present a type I-D (TiD) CRISPR-Cas genome editing system in plants. TiD lacks the Cas3 nuclease domain; instead, Cas10d is the functional nuclease in vivo. TiD was active in targeted mutagenesis of tomato genomic DNA. The mutations generated by TiD differed from those of CRISPR/Cas9; both bi-directional long-range deletions and short indels mutations were detected in tomato cells. Furthermore, TiD can be used to efficiently generate bi-allelic mutant plants in the first generation. These findings indicate that TiD is a unique CRISPR system that can be used for genome engineering in plants.

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Genome Editing Technologies: From Bench Side to Bedside

Acta Medica ◽

10.32552/2018.actamedica.282 ◽

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.

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CRISPR/Cpf1 mediated genome editing enhances Bombyx mori resistance to BmNPV

10.1101/2020.03.23.003368 ◽

2020 ◽

Author(s):

Zhanqi Dong ◽

Qi Qin ◽

Zhigang Hu ◽

Xinling Zhang ◽

Jianghao Miao ◽

...

Keyword(s):

Antiviral Therapy ◽

Genome Editing ◽

Genome Engineering ◽

Transgenic Silkworm ◽

Editing Efficiency ◽

Transgenic Lines ◽

Transgenic Silkworms ◽

Bmnpv Genome

AbstractCRISPR/Cas12a (Cpf1) is a single RNA-guided endonuclease that provides new opportunities for targeted genome engineering through the CRISPR/Cas9 system. Only AsCpf1 have been developed for insect genome editing, and the novel Cas12a orthologs nucleases and editing efficiency require more study in insect. We compared three Cas12a orthologs nucleases, AsCpf1, FnCpf1, and LbCpf1, for their editing efficiencies and antiviral abilities in vitro. The three Cpf1 efficiently edited the BmNPV genome and inhibited BmNPV replication in BmN-SWU1 cells. The antiviral ability of the FnCpf1 system was more efficient than the SpCas9 system after infection by BmNPV. We created FnCpf1×gIE1 and SpCas9×sgIE1 transgenic hybrid lines and evaluated the gene editing efficiency of different systems at the same target site. We improved the antiviral ability using the FnCpf1 system in transgenic silkworm. This study demonstrated use of the CRISPR/Cpf1 system to achieve high editing efficiencies in the silkworm, and illustrates the use of this technology for increasing disease resistance.Author SummaryGenome editing is a powerful tool that has been widely used in gene function, gene therapy, pest control, and disease-resistant engineering in most parts of pathogens research. Since the establishment of CRISPR/Cas9, powerful strategies for antiviral therapy of transgenic silkworm have emerged. Nevertheless, there is still room to expand the scope of genome editing tool for further application to improve antiviral research. Here, we demonstrate that three Cpf1 endonuclease can be used efficiency editing BmNPV genome in vitro and in vivo for the first time. More importantly, this Cpf1 system could improve the resistance of transgenic silkworms to BmNPV compare with Cas9 system, and no significant cocoons difference was observed between transgenic lines infected with BmNPV and control. These broaden the range of application of CRISPR for novel genome editing methods in silkworm and also enable sheds light on antiviral therapy.

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Integrating Biomaterials and Genome Editing Approaches to Advance Biomedical Science

Annual Review of Biomedical Engineering ◽

10.1146/annurev-bioeng-122019-121602 ◽

2021 ◽

Vol 23 (1) ◽

Author(s):

Amr A. Abdeen ◽

Brian D. Cosgrove ◽

Charles A. Gersbach ◽

Krishanu Saha

Keyword(s):

Genome Editing ◽

Gene Editing ◽

Genome Engineering ◽

Subsequent Development ◽

Biomedical Science ◽

Annual Review ◽

Publication Date ◽

Nonviral Delivery ◽

Short Palindromic Repeat

The recent discovery and subsequent development of the clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated (Cas) platform as a precise genome editing tool have transformed biomedicine. As these CRISPR-based tools have matured, multiple stages of the gene editing process and the bioengineering of human cells and tissues have advanced. Here, we highlight recent intersections in the development of biomaterials and genome editing technologies. These intersections include the delivery of macromolecules, where biomaterial platforms have been harnessed to enable nonviral delivery of genome engineering tools to cells and tissues in vivo. Further, engineering native-like biomaterial platforms for cell culture facilitates complex modeling of human development and disease when combined with genome engineering tools. Deeper integration of biomaterial platforms in these fields could play a significant role in enabling new breakthroughs in the application of gene editing for the treatment of human disease. Expected final online publication date for the Annual Review of Biomedical Engineering, Volume 23 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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Targeted editing and evolution of engineered ribosomes in vivo by filtered editing

Nature Communications ◽

10.1038/s41467-021-27836-x ◽

2022 ◽

Vol 13 (1) ◽

Author(s):

Felix Radford ◽

Shane D. Elliott ◽

Alanna Schepartz ◽

Farren J. Isaacs

Keyword(s):

Genome Editing ◽

Dna Sequences ◽

Genome Engineering ◽

23S Rrna ◽

Translational Efficiency ◽

Rna Molecules ◽

Genetic Elements ◽

A Genome ◽

Self Splicing

AbstractGenome editing technologies introduce targeted chromosomal modifications in organisms yet are constrained by the inability to selectively modify repetitive genetic elements. Here we describe filtered editing, a genome editing method that embeds group 1 self-splicing introns into repetitive genetic elements to construct unique genetic addresses that can be selectively modified. We introduce intron-containing ribosomes into the E. coli genome and perform targeted modifications of these ribosomes using CRISPR/Cas9 and multiplex automated genome engineering. Self-splicing of introns post-transcription yields scarless RNA molecules, generating a complex library of targeted combinatorial variants. We use filtered editing to co-evolve the 16S rRNA to tune the ribosome’s translational efficiency and the 23S rRNA to isolate antibiotic-resistant ribosome variants without interfering with native translation. This work sets the stage to engineer mutant ribosomes that polymerize abiological monomers with diverse chemistries and expands the scope of genome engineering for precise editing and evolution of repetitive DNA sequences.

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Annexin Animal Models—From Fundamental Principles to Translational Research

International Journal of Molecular Sciences ◽

10.3390/ijms22073439 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3439

Author(s):

Thomas Grewal ◽

Carles Rentero ◽

Carlos Enrich ◽

Mohamed Wahba ◽

Carsten A. Raabe ◽

...

Keyword(s):

Animal Models ◽

Mouse Models ◽

Developmental Stages ◽

Membrane Lipids ◽

Living Organism ◽

Lipid Binding ◽

Experimental Medicine ◽

Comprehensive Overview

Routine manipulation of the mouse genome has become a landmark in biomedical research. Traits that are only associated with advanced developmental stages can now be investigated within a living organism, and the in vivo analysis of corresponding phenotypes and functions advances the translation into the clinical setting. The annexins, a family of closely related calcium (Ca2+)- and lipid-binding proteins, are found at various intra- and extracellular locations, and interact with a broad range of membrane lipids and proteins. Their impacts on cellular functions has been extensively assessed in vitro, yet annexin-deficient mouse models generally develop normally and do not display obvious phenotypes. Only in recent years, studies examining genetically modified annexin mouse models which were exposed to stress conditions mimicking human disease often revealed striking phenotypes. This review is the first comprehensive overview of annexin-related research using animal models and their exciting future use for relevant issues in biology and experimental medicine.

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