scholarly journals CRISPR/Cas9 gene-editing strategies in cardiovascular cells

2019 ◽  
Vol 116 (5) ◽  
pp. 894-907 ◽  
Author(s):  
Eva Vermersch ◽  
Charlène Jouve ◽  
Jean-Sébastien Hulot
Keyword(s):  
Cardiovascular Diseases ◽  
Genome Editing ◽  
Zinc Finger Nucleases ◽  
Public Health Issue ◽  
Transcription Activator ◽  
Knock Out ◽  
Cardiovascular Cells ◽  
Induced Pluripotent

Abstract Cardiovascular diseases are among the main causes of morbidity and mortality in Western countries and considered as a leading public health issue. Therefore, there is a strong need for new disease models to support the development of novel therapeutics approaches. The successive improvement of genome editing tools with zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and more recently with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) has enabled the generation of genetically modified cells and organisms with much greater efficiency and precision than before. The simplicity of CRISPR/Cas9 technology made it especially suited for different studies, both in vitro and in vivo, and has been used in multiple studies evaluating gene functions, disease modelling, transcriptional regulation, and testing of novel therapeutic approaches. Notably, with the parallel development of human induced pluripotent stem cells (hiPSCs), the generation of knock-out and knock-in human cell lines significantly increased our understanding of mutation impacts and physiopathological mechanisms within the cardiovascular domain. Here, we review the recent development of CRISPR–Cas9 genome editing, the alternative tools, the available strategies to conduct genome editing in cardiovascular cells with a focus on its use for correcting mutations in vitro and in vivo both in germ and somatic cells. We will also highlight that, despite its potential, CRISPR/Cas9 technology comes with important technical and ethical limitations. The development of CRISPR/Cas9 genome editing for cardiovascular diseases indeed requires to develop a specific strategy in order to optimize the design of the genome editing tools, the manipulation of DNA repair mechanisms, the packaging and delivery of the tools to the studied organism, and the assessment of their efficiency and safety.

scholarly journals Efficient in vivo genome editing mediated by stem cells-derived extracellular vesicles carrying designer nucleases

2021 ◽  
Author(s):  
Sylwia Bobis-Wozowicz ◽  
Karolina Kania ◽  
Kinga Nit ◽  
Natalia Blazowska ◽  
Katarzyna Kmiotek-Wasylewska ◽  
...  
Keyword(s):  
Stem Cells ◽  
Genome Editing ◽  
Extracellular Vesicles ◽  
Gene Knockout ◽  
Marker Gene ◽  
Cell Types ◽  
Zinc Finger Nucleases ◽  
Transcription Activator

Precise genome editing using designer nucleases (DNs), such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system, has become a method of choice in a variety of biological and biomedical applications in recent years. Notably, efficacy of these systems is currently under scrutiny in about 50 clinical trials. Although high DNs activity in various cell types in vitro has already been achieved, efficient in vivo genome editing remains a challenge. To solve this problem, we employed stem cells-derived extracellular vesicles (EVs) as carriers of DNs. We used umbilical cord-derived mesenchymal stem cells (UC-MSCs) and induced pluripotent stem cells (iPSCs) as EV-producer cells, since they are both applied in regenerative medicine. In our proof-of-concept studies, we achieved up to 50% of EGFP marker gene knockout in vivo using EVs carrying either ZFN, TALEN or the CRISPR/Cas9 system, particularly in the liver. Importantly, we obtained almost 50% of modified alleles in the liver of the experimental animals, when targeting the Pcsk9 gene, whose overexpression is implicated in hypercholesterolemia. Taken together, our data provide strong evidence that stem cells-derived EVs constitute a robust tool in delivering DNs in vivo, which may be harnessed to clinical practice in the future.

31 PRODUCTION EFFICIENCY OF GENE KNOCKOUT PIGS USING GENOME EDITING AND SOMATIC CELL CLONING

2015 ◽  
Vol 27 (1) ◽  
pp. 108
Author(s):  
H. Matsunari ◽  
M. Watanabe ◽  
K. Nakano ◽  
A. Uchikura ◽  
Y. Asano ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Genome Editing ◽  
Production Efficiency ◽  
Gene Knockout ◽  
Genetically Modified ◽  
Zinc Finger Nucleases ◽  
International Institute ◽  
Induced Mutations ◽  
Transcription Activator

Genome editing technologies have been used as a powerful strategy for the generation of genetically modified pigs. We previously developed genetically modified clone pigs with organogenesis-disabled phenotypes, as well as pigs exhibiting diseases with similar features to those of humans. Here, we report the production efficiency of various gene knockout cloned pigs from somatic cells that were genetically modified using zinc finger nucleases (ZFN) or transcription activator-like effector nucleases (TALEN). The ZFN- or TALEN-encoding mRNAs, which targeted 7 autosomal or X-linked genes, were introduced into porcine fetal fibroblast cells using electroporation. Clonal cell populations carrying induced mutations were selected after limiting dilution. The targeted portion of the genes was amplified using PCR, followed by sequencing and mutation analysis. Among the collected knockout cell colonies, cells showing good proliferation and morphology were selected and used for somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were obtained from porcine cumulus-oocyte complexes cultured in NCSU23-based medium and were used to obtain recipient oocytes for SCNT after enucleation. SCNT was performed as reported previously (Matsunari et al. 2008). The cloned embryos were cultured for 7 days in porcine zygote medium (PZM)-5 to assess their developmental ability. Cloned embryos were transplanted into the oviduct or uterus of oestrus-synchronized recipient gilts to evaluate their competence to develop to fetuses or piglets. Cloned embryos reconstructed with 7 types of knockout cells showed equal development to blastocysts compared with those derived from the wild-type cells (54.5–83.3% v. 60.7%). Our data (Table 1) demonstrated that the reconstructed embryos derived from knockout cells could efficiently give rise to cloned offspring regardless of the type of genome editing methodology (i.e. ZFN or TALEN). Table 1.Production efficiency of gene knockout cloned pigs using genome editing This study was supported by JST, ERATO, the Nakauchi Stem Cell and Organ Regeneration Project, JST, CREST, Meiji University International Institute for Bio-Resource Research (MUIIBR), and JSPS KAKENHI Grant Number 26870630.

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.

T-Cell Engineering For “off-The-shelf” Adoptive Immunotherapy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1661-1661
Author(s):  
Laurent Poirot ◽  
Cécile Schiffer-Mannioui ◽  
Brian Philip ◽  
Sophie Derniame ◽  
Agnes Gouble ◽  
...  
Keyword(s):  
T Cells ◽  
United Kingdom ◽  
T Cell ◽  
Adoptive Immunotherapy ◽  
Functional Characterization ◽  
Immunosuppressive Drug ◽  
Transcription Activator ◽  
Knock Out

Abstract Adoptive T-cell therapies, where exogenous expression of a chimeric antigen receptor (CAR) confers cancer recognition, have shown significant promise in initial clinical trials. However, present adoptive immunotherapy Methods are limited by the need for manipulation of autologous patient T-cells. To permit such an approach in an allogeneic context, Transcription Activator-Like Effector Nucleases (TALENTM) have been used to simultaneously inactivate the endogenous T cell receptor and CD52, a cellular target for a lymphodepleting treatment. This approach reduces the risk of GVHD while permitting proliferation and activity of the introduced T lymphocytes in the presence of the immunosuppressive drug alemtuzumab. Electroporation of primary T cells with mRNA coding for the appropriate TALENTM result in double knock-out (dKO) frequencies of up to 70%. Furthermore, functional characterization demonstrates that the dKO cells are resistant to complement dependent lysis or in vivo depletion by alemtuzumab, and show no apparent potential for TCR-mediated activation. Finally, endowing the dKO cells with a CD19 CAR supports their capacity to kill CD19+ tumor targets as efficiently as unedited T-cells both in vitro and in vivo. Disclosures: Poirot: CELLECTIS THERAPEUTICS: Employment. Schiffer-Mannioui:CELLECTIS THERAPEUTICS: Employment. Philip:UCL Cancer Institute, London, United Kingdom: Employment. Derniame:CELLECTIS THERAPEUTICS: Employment. Gouble:CELLECTIS THERAPEUTICS: Employment. Chion-Sotinel:CELLECTIS THERAPEUTICS: Employment. Le Clerre:CELLECTIS THERAPEUTICS: Employment. Lemaire:CELLECTIS THERAPEUTICS: Employment. Grosse:CELLECTIS THERAPEUTICS: Employment. Cheung:UCL Cancer Institute, London, United Kingdom: Employment. Arnould:CELLECTIS THERAPEUTICS: Employment. Smith:CELLECTIS THERAPEUTICS: Employment. Pule:UCL Cancer Institute, London, United Kingdom: Employment. Scharenberg:CELLECTIS THERAPEUTICS: Employment.

scholarly journals The Impact of CRISPR/Cas9 Technology on Cardiac Research: From Disease Modelling to Therapeutic Approaches

2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
Benedetta M. Motta ◽  
Peter P. Pramstaller ◽  
Andrew A. Hicks ◽  
Alessandra Rossini
Keyword(s):  
Genome Editing ◽  
Ethical Issues ◽  
Therapeutic Potential ◽  
Gene Knockout ◽  
Model Systems ◽  
Large Deletions ◽  
Induced Pluripotent ◽  
The Impact

Genome-editing technology has emerged as a powerful method that enables the generation of genetically modified cells and organisms necessary to elucidate gene function and mechanisms of human diseases. The clustered regularly interspaced short palindromic repeats- (CRISPR-) associated 9 (Cas9) system has rapidly become one of the most popular approaches for genome editing in basic biomedical research over recent years because of its simplicity and adaptability. CRISPR/Cas9 genome editing has been used to correct DNA mutations ranging from a single base pair to large deletions in both in vitro and in vivo model systems. CRISPR/Cas9 has been used to increase the understanding of many aspects of cardiovascular disorders, including lipid metabolism, electrophysiology and genetic inheritance. The CRISPR/Cas9 technology has been proven to be effective in creating gene knockout (KO) or knockin in human cells and is particularly useful for editing induced pluripotent stem cells (iPSCs). Despite these progresses, some biological, technical, and ethical issues are limiting the therapeutic potential of genome editing in cardiovascular diseases. This review will focus on various applications of CRISPR/Cas9 genome editing in the cardiovascular field, for both disease research and the prospect of in vivo genome-editing therapies in the future.

CRISPER/CAS: A potential tool for genomes editing

2021 ◽  
Vol 7 (2) ◽  
pp. 122-129
Keyword(s):  
Genome Editing ◽  
Basic Mechanism ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
End Joining ◽  
Knock Out ◽  
Homology Directed Repair ◽  
Wide Range ◽  
Non Homologous End Joining ◽  
Cas A

The ability to engineer genomes presents a significant opportunity for applied biology research. In 2050, the population of this world is expected to reach 9.6 billion residents; rising food with better quality is the most promising approach to food security. Compared to earlier methodologies including Zinc Finger Nucleases (ZFNs) plus Transcription Activator-Like Effector Nucleases (TALENs), which were expensive as well as time-consuming, innovation in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and related CRISPR (Cas) protein classifications allowed selective editing of genes for the enhancement of food. The basic mechanism of CRISPR Cas9 process and its applications on genome editing has been summarized in this manuscript. The method relies on Sequence-Specific Nucleases (SSNs) to create Double Stranded Breaks (DSB) of DNA at the locus of genome defined by user, mended by using one of two DNA mending ways: Non-Homologous End Joining (NHEJ) or Homology Directed Repair (HDR). Cas9, an RNA-guided endonuclease, was used to produce stable knock-in and knock-out mutants. The focus of this effort is to explore the CRISPR Cas9 genome editing to manage gene expression and improve future editing success. This adaptable technique can be consumed for a wide range of applications of genome editing requiring high precision. Advances in this technology have sparked renewed interest in the possibilities for editing genome in plants.

scholarly journals A Newly Built rAAV-SaCas9 Genome Editing System Enables Muscle-Directed Gene Editing to Improve Muscular Atrophy

2020 ◽  
Author(s):  
Shaoting Weng ◽  
Xingyu Li ◽  
Yitian Zhao ◽  
Feng Gao ◽  
Mengmeng Shi ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Fluorescent Protein ◽  
Therapeutic Potential ◽  
Muscular Atrophy ◽  
Virus Titer ◽  
Myostatin Gene ◽  
Knock Out

Abstract Background At present, genome editing at specific sites in vivo is affected by many factors, including the choice of vector, the efficiency of editing proteins and the influence of the internal environment. These factors make gene editing ineffective and even have adverse effects. Methods Here, we report a single rAAV containing SaCas9 and guide RNAs under the control of subtle EF1a and tRNA promoters. The capacity of rAAV was compressed, and we inserted the sequence of the green fluorescent protein eGFP into rAAV. The efficiency of rAAV gene editing in vivo and in vitro was analyzed by time point and virus titer. In addition, we used the rAAV9-SaCas9 system to knock out the myostatin gene in the thigh muscles of muscle-atrophic mice. Results We demonstrated that the gene editing elements regulated by the rAAV-SaCas9 system can be expressed. By increasing the amount of rAAV and the reaction time, the editing efficiency of myostatin and the expression level of eGFP protein can be improved in vitro and vivo. Furthermore, We demonstrated that muscle cells were improved by knockout partial myostatin gene in a mouse model of muscular dystrophy. Conclusions The rAAV-SaCas9 system can be expressed in a stable and long-term manner. The system has substantial therapeutic potential in treating muscular atrophy.

scholarly journals In Vivo Genome Editing as a Therapeutic Approach

2018 ◽  
Vol 19 (9) ◽  
pp. 2721 ◽  
Author(s):  
Beatrice Ho ◽  
Sharon Loh ◽  
Woon Chan ◽  
Boon Soh
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Genetic Diseases ◽  
Gene Mutations ◽  
Genetic Mutation ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Therapeutic Benefits ◽  
A Genome

Genome editing has been well established as a genome engineering tool that enables researchers to establish causal linkages between genetic mutation and biological phenotypes, providing further understanding of the genetic manifestation of many debilitating diseases. More recently, the paradigm of genome editing technologies has evolved to include the correction of mutations that cause diseases via the use of nucleases such as zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and more recently, Cas9 nuclease. With the aim of reversing disease phenotypes, which arise from somatic gene mutations, current research focuses on the clinical translatability of correcting human genetic diseases in vivo, to provide long-term therapeutic benefits and potentially circumvent the limitations of in vivo cell replacement therapy. In this review, in addition to providing an overview of the various genome editing techniques available, we have also summarized several in vivo genome engineering strategies that have successfully demonstrated disease correction via in vivo genome editing. The various benefits and challenges faced in applying in vivo genome editing in humans will also be discussed.

scholarly journals CRISPR/Cas Derivatives as Novel Gene Modulating Tools: Possibilities and In Vivo Applications

2020 ◽  
Vol 21 (9) ◽  
pp. 3038 ◽  
Author(s):  
Xingbo Xu ◽  
Melanie S. Hulshoff ◽  
Xiaoying Tan ◽  
Michael Zeisberg ◽  
Elisabeth M. Zeisberg
Keyword(s):  
Transcriptional Activation ◽  
Gene Activation ◽  
Zinc Finger Nucleases ◽  
Homing Endonucleases ◽  
Transcription Activator ◽  
Base Editing ◽  
Advantages And Disadvantages ◽  
Associated Proteins

The field of genome editing started with the discovery of meganucleases (e.g., the LAGLIDADG family of homing endonucleases) in yeast. After the discovery of transcription activator-like effector nucleases and zinc finger nucleases, the recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) system has opened a new window of applications in the field of gene editing. Here, we review different Cas proteins and their corresponding features including advantages and disadvantages, and we provide an overview of the different endonuclease-deficient Cas protein (dCas) derivatives. These dCas derivatives consist of an endonuclease-deficient Cas9 which can be fused to different effector domains to perform distinct in vitro applications such as tracking, transcriptional activation and repression, as well as base editing. Finally, we review the in vivo applications of these dCas derivatives and discuss their potential to perform gene activation and repression in vivo, as well as their potential future use in human therapy.

scholarly journals INDEL detection, the ‘Achilles heel’ of precise genome editing: a survey of methods for accurate profiling of gene editing induced indels

2020 ◽  
Vol 48 (21) ◽  
pp. 11958-11981
Author(s):  
Eric Paul Bennett ◽  
Bent Larsen Petersen ◽  
Ida Elisabeth Johansen ◽  
Yiyuan Niu ◽  
Zhang Yang ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Strand Break ◽  
Zinc Finger Nucleases ◽  
Great Promise ◽  
Transcription Activator ◽  
End Joining ◽  
Knock Out ◽  
Indel Detection ◽  
In Cells

Abstract Advances in genome editing technologies have enabled manipulation of genomes at the single base level. These technologies are based on programmable nucleases (PNs) that include meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) nucleases and have given researchers the ability to delete, insert or replace genomic DNA in cells, tissues and whole organisms. The great flexibility in re-designing the genomic target specificity of PNs has vastly expanded the scope of gene editing applications in life science, and shows great promise for development of the next generation gene therapies. PN technologies share the principle of inducing a DNA double-strand break (DSB) at a user-specified site in the genome, followed by cellular repair of the induced DSB. PN-elicited DSBs are mainly repaired by the non-homologous end joining (NHEJ) and the microhomology-mediated end joining (MMEJ) pathways, which can elicit a variety of small insertion or deletion (indel) mutations. If indels are elicited in a protein coding sequence and shift the reading frame, targeted gene knock out (KO) can readily be achieved using either of the available PNs. Despite the ease by which gene inactivation in principle can be achieved, in practice, successful KO is not only determined by the efficiency of NHEJ and MMEJ repair; it also depends on the design and properties of the PN utilized, delivery format chosen, the preferred indel repair outcomes at the targeted site, the chromatin state of the target site and the relative activities of the repair pathways in the edited cells. These variables preclude accurate prediction of the nature and frequency of PN induced indels. A key step of any gene KO experiment therefore becomes the detection, characterization and quantification of the indel(s) induced at the targeted genomic site in cells, tissues or whole organisms. In this survey, we briefly review naturally occurring indels and their detection. Next, we review the methods that have been developed for detection of PN-induced indels. We briefly outline the experimental steps and describe the pros and cons of the various methods to help users decide a suitable method for their editing application. We highlight recent advances that enable accurate and sensitive quantification of indel events in cells regardless of their genome complexity, turning a complex pool of different indel events into informative indel profiles. Finally, we review what has been learned about PN-elicited indel formation through the use of the new methods and how this insight is helping to further advance the genome editing field.

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