From Descriptive to Functional Genomics of Leukemias Focusing on Genome Engineering Techniques

Genome engineering makes the precise manipulation of DNA sequences possible in a cell. Therefore, it is essential for understanding gene function. Meganucleases were the start of genome engineering, and it continued with the discovery of Zinc finger nucleases (ZFNs), followed by Transcription activator-like effector nucleases (TALENs). They can generate double-strand breaks at a desired target site in the genome, and therefore can be used to knock in mutations or knock out genes in the same way. Years later, genome engineering was transformed by the discovery of clustered regularly interspaced short palindromic repeats (CRISPR). Implementation of CRISPR systems involves recognition guided by RNA and the precise cleaving of DNA molecules. This property proves its utility in epigenetics and genome engineering. CRISPR has been and is being continuously successfully used to model mutations in leukemic cell lines and control gene expression. Furthermore, it is used to identify targets and discover drugs for immune therapies. The descriptive and functional genomics of leukemias is discussed in this study, with an emphasis on genome engineering methods. The CRISPR/Cas9 system’s challenges, viewpoints, limits, and solutions are also explored.

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Advances in methods for reducing mitochondrial DNA disease by replacing or manipulating the mitochondrial genome

Essays in Biochemistry ◽

10.1042/ebc20170113 ◽

2018 ◽

Vol 62 (3) ◽

pp. 455-465 ◽

Cited By ~ 16

Author(s):

Pavandeep K. Rai ◽

Lyndsey Craven ◽

Kurt Hoogewijs ◽

Oliver M. Russell ◽

Robert N. Lightowlers

Keyword(s):

Mitochondrial Dna ◽

Mitochondrial Genome ◽

Genome Engineering ◽

Wide Spectrum ◽

Tissue Type ◽

Zinc Finger Nucleases ◽

Transcription Activator ◽

Mitochondrial Donation ◽

A Cell ◽

Maternal Spindle Transfer

Mitochondrial DNA (mtDNA) is a multi-copy genome whose cell copy number varies depending on tissue type. Mutations in mtDNA can cause a wide spectrum of diseases. Mutated mtDNA is often found as a subset of the total mtDNA population in a cell or tissue, a situation known as heteroplasmy. As mitochondrial dysfunction only presents after a certain level of heteroplasmy has been acquired, ways to artificially reduce or replace the mutated species have been attempted. This review addresses recent approaches and advances in this field, focusing on the prevention of pathogenic mtDNA transfer via mitochondrial donation techniques such as maternal spindle transfer and pronuclear transfer in which mutated mtDNA in the oocyte or fertilized embryo is substituted with normal copies of the mitochondrial genome. This review also discusses the molecular targeting and cleavage of pathogenic mtDNA to shift heteroplasmy using antigenomic therapy and genome engineering techniques including Zinc-finger nucleases and transcription activator-like effector nucleases. Finally, it considers CRISPR technology and the unique difficulties that mitochondrial genome editing presents.

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TALEN and CRISPR/Cas Genome Editing Systems: Tools of Discovery

Acta Naturae ◽

10.32607/20758251-2014-6-3-19-40 ◽

2014 ◽

Vol 6 (3) ◽

pp. 19-40 ◽

Cited By ~ 94

Author(s):

A. A. Nemudryi ◽

K. R. Valetdinova ◽

S. P. Medvedev ◽

S. M. Zakian

Keyword(s):

Genome Editing ◽

Dna Sequences ◽

Genome Engineering ◽

Disease Modeling ◽

Regulation Of Gene Expression ◽

Hereditary Disease ◽

Transcription Activator ◽

Wide Range ◽

High Standards ◽

Phenotype Formation

Precise studies of plant, animal and human genomes enable remarkable opportunities of obtained data application in biotechnology and medicine. However, knowing nucleotide sequences isnt enough for understanding of particular genomic elements functional relationship and their role in phenotype formation and disease pathogenesis. In post-genomic era methods allowing genomic DNA sequences manipulation, visualization and regulation of gene expression are rapidly evolving. Though, there are few methods, that meet high standards of efficiency, safety and accessibility for a wide range of researchers. In 2011 and 2013 novel methods of genome editing appeared - this are TALEN (Transcription Activator-Like Effector Nucleases) and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems. Although TALEN and CRISPR/Cas9 appeared recently, these systems have proved to be effective and reliable tools for genome engineering. Here we generally review application of these systems for genome editing in conventional model objects of current biology, functional genome screening, cell-based human hereditary disease modeling, epigenome studies and visualization of cellular processes. Additionally, we review general strategies for designing TALEN and CRISPR/Cas9 and analyzing their activity. We also discuss some obstacles researcher can face using these genome editing tools.

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Genome Editing Toolbox

Blood ◽

10.1182/blood.v124.21.sci-11.sci-11 ◽

2014 ◽

Vol 124 (21) ◽

pp. SCI-11-SCI-11

Author(s):

Andrew M. Scharenberg

Keyword(s):

Genome Engineering ◽

Gene Knockout ◽

Zinc Finger Nucleases ◽

Practical Implementation ◽

Homing Endonucleases ◽

Gene Repair ◽

Dna Breaks ◽

Transcription Activator ◽

Advisory Committees ◽

Core Technology

Abstract Nucleases capable of making targeted breaks in genomic DNA are a core technology required for genome engineering, an emerging field of technology for making precise alterations in cellular genomes. Over the past ten years, four major platforms have emerged for generation of nucleases able to make targeted DNA breaks with a high degree of efficiency and specificity: homing endonucleases, zinc finger nucleases, transcription activator-like (TAL) effector nucleases, and RNA-guided nucleases. This talk will cover the biochemistry and platform-specific attributes of each type of nuclease, along with evolution/improvements in nucleases and related technologies and aspects of the practical implementation of nuclease technology for gene knockout and gene repair in primary hematopoietic cells. Disclosures Scharenberg: Pregenen Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cellectis therapeutics: Consultancy.

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Modern Trends in Plant Genome Editing: An Inclusive Review of the CRISPR/Cas9 Toolbox

International Journal of Molecular Sciences ◽

10.3390/ijms20164045 ◽

2019 ◽

Vol 20 (16) ◽

pp. 4045 ◽

Cited By ~ 14

Author(s):

Ali Razzaq ◽

Fozia Saleem ◽

Mehak Kanwal ◽

Ghulam Mustafa ◽

Sumaira Yousaf ◽

...

Keyword(s):

Genome Editing ◽

Genome Engineering ◽

Crop Improvement ◽

Crop Plants ◽

Targeted Mutagenesis ◽

Plant Genome ◽

Zinc Finger Nucleases ◽

Base Substitution ◽

Transcription Activator ◽

Abiotic Stressors

Increasing agricultural productivity via modern breeding strategies is of prime interest to attain global food security. An array of biotic and abiotic stressors affect productivity as well as the quality of crop plants, and it is a primary need to develop crops with improved adaptability, high productivity, and resilience against these biotic/abiotic stressors. Conventional approaches to genetic engineering involve tedious procedures. State-of-the-art OMICS approaches reinforced with next-generation sequencing and the latest developments in genome editing tools have paved the way for targeted mutagenesis, opening new horizons for precise genome engineering. Various genome editing tools such as transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and meganucleases (MNs) have enabled plant scientists to manipulate desired genes in crop plants. However, these approaches are expensive and laborious involving complex procedures for successful editing. Conversely, CRISPR/Cas9 is an entrancing, easy-to-design, cost-effective, and versatile tool for precise and efficient plant genome editing. In recent years, the CRISPR/Cas9 system has emerged as a powerful tool for targeted mutagenesis, including single base substitution, multiplex gene editing, gene knockouts, and regulation of gene transcription in plants. Thus, CRISPR/Cas9-based genome editing has demonstrated great potential for crop improvement but regulation of genome-edited crops is still in its infancy. Here, we extensively reviewed the availability of CRISPR/Cas9 genome editing tools for plant biotechnologists to target desired genes and its vast applications in crop breeding research.

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Self-cutting and integrating CRISPR plasmids (SCIPs) enable targeted genomic integration of genetic payloads for rapid cell engineering

10.1101/2020.03.25.008276 ◽

2020 ◽

Author(s):

Darin Bloemberg ◽

Daniela Sosa-Miranda ◽

Tina Nguyen ◽

Risini D. Weeratna ◽

Scott McComb

Keyword(s):

T Cell Activation ◽

Mammalian Cells ◽

Cell Activation ◽

Genome Engineering ◽

Jurkat Cells ◽

Sequencing Analysis ◽

Knock Out ◽

Homology Directed Repair ◽

A Cell ◽

Future Utility

AbstractSince observations that CRISPR nucleases function in mammalian cells, many strategies have been devised to adapt them for genetic engineering. Here, we investigated self-cutting and integrating CRISPR-Cas9 plasmids (SCIPs) as easy-to-use gene editing tools that insert themselves at CRISPR-guided locations. SCIPs demonstrated similar expression kinetics and gene disruption efficiency in mouse (EL4) and human (Jurkat) cells, with stable integration in 3-6% of transfected cells. Clonal sequencing analysis indicated that integrants showed bi- or mono-allelic integration of entire CRISPR plasmids in predictable orientations and with limited indel formation. Interestingly, including longer homology arms (HAs) (500 bp) in varying orientations only modestly increased knock-in efficiency (∼2-fold). Using a SCIP-payload design (SCIPpay) which liberates a promoter-less sequence flanked by HAs thereby requiring perfect homology-directed repair (HDR) for transgene expression, longer HAs resulted in higher integration efficiency and precision of the payload but did not affect integration of the remaining plasmid sequence. As proofs-of-concept, we used SCIPpay to 1) insert a gene fragment encoding tdTomato into the CD69 locus of Jurkat cells, thereby creating a cell line that reports T cell activation, and 2) insert a chimeric antigen receptor (CAR) gene into the TRAC locus. Here, we demonstrate that SCIPs function as simple, efficient, and programmable tools useful for generating gene knock-out/knock-in cell lines and suggest future utility in knock-in site screening/optimization, unbiased off-target site identification, and multiplexed, iterative, and/or library-scale automated genome engineering.

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Gene editing and CRISPR in the clinic: current and future perspectives

Bioscience Reports ◽

10.1042/bsr20200127 ◽

2020 ◽

Vol 40 (4) ◽

Cited By ~ 16

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.

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TALENs—an indispensable tool in the era of CRISPR: a mini review

Journal of Genetic Engineering and Biotechnology ◽

10.1186/s43141-021-00225-z ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Anuradha Bhardwaj ◽

Vikrant Nain

Keyword(s):

Genome Editing ◽

Genome Engineering ◽

High Specificity ◽

Zinc Finger Nucleases ◽

Main Body ◽

Transcription Activator ◽

Scientific World ◽

Dna Modifications ◽

Pros And Cons ◽

Indispensable Tool

Abstract Background Genome of an organism has always fascinated life scientists. With the discovery of restriction endonucleases, scientists were able to make targeted manipulations (knockouts) in any gene sequence of any organism, by the technique popularly known as genome engineering. Though there is a range of genome editing tools, but this era of genome editing is dominated by the CRISPR/Cas9 tool due to its ease of design and handling. But, when it comes to clinical applications, CRISPR is not usually preferred. In this review, we will elaborate on the structural and functional role of designer nucleases with emphasis on TALENs and CRISPR/Cas9 genome editing system. We will also present the unique features of TALENs and limitations of CRISPRs which makes TALENs a better genome editing tool than CRISPRs. Main body Genome editing is a robust technology used to make target specific DNA modifications in the genome of any organism. With the discovery of robust programmable endonucleases-based designer gene manipulating tools such as meganucleases (MN), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats associated protein (CRISPR/Cas9), the research in this field has experienced a tremendous acceleration giving rise to a modern era of genome editing with better precision and specificity. Though, CRISPR-Cas9 platform has successfully gained more attention in the scientific world, TALENs and ZFNs are unique in their own ways. Apart from high-specificity, TALENs are proven to target the mitochondrial DNA (mito-TALEN), where gRNA of CRISPR is difficult to import. This review talks about genome editing goals fulfilled by TALENs and drawbacks of CRISPRs. Conclusions This review provides significant insights into the pros and cons of the two most popular genome editing tools TALENs and CRISPRs. This mini review suggests that, TALENs provides novel opportunities in the field of therapeutics being highly specific and sensitive toward DNA modifications. In this article, we will briefly explore the special features of TALENs that makes this tool indispensable in the field of synthetic biology. This mini review provides great perspective in providing true guidance to the researchers working in the field of trait improvement via genome editing.

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Rescuing Lethal Phenotypes Induced by Disruption of Genes in Mice: a Review of Novel Strategies

Physiological Research ◽

10.33549/physiolres.934543 ◽

2021 ◽

pp. 3-12

Author(s):

N LIPTÁK ◽

Z GÁL ◽

B BIRÓ ◽

L HIRIPI ◽

O HOFFMANN

Keyword(s):

Human Disease ◽

Mendelian Inheritance ◽

Zinc Finger Nucleases ◽

Specific Gene ◽

Transcription Activator ◽

Knock Out ◽

Developmental Abnormalities ◽

Manual Search ◽

Cas9 Nuclease ◽

Omim Database

Approximately 35 % of the mouse genes are indispensable for life, thus, global knock-out (KO) of those genes may result in embryonic or early postnatal lethality due to developmental abnormalities. Several KO mouse lines are valuable human disease models, but viable homozygous mutant mice are frequently required to mirror most symptoms of a human disease. The site-specific gene editing systems, the transcription activator-like effector nucleases (TALENs), Zinc-finger nucleases (ZFNs) and the clustered regularly interspaced short palindrome repeat-associated Cas9 nuclease (CRISPR/Cas9) made the generation of KO mice more efficient than before, but the homozygous lethality is still an undesired side-effect in case of many genes. The literature search was conducted using PubMed and Web of Science databases until June 30th, 2020. The following terms were combined to find relevant studies: “lethality”, “mice”, “knock-out”, “deficient”, “embryonic”, “perinatal”, “rescue”. Additional manual search was also performed to find the related human diseases in the Online Mendelian Inheritance in Man (OMIM) database and to check the citations of the selected studies for rescuing methods. In this review, the possible solutions for rescuing human disease-relevant homozygous KO mice lethal phenotypes were summarized.

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Empowering of reproductive health of farm animals through genome editing technology

10.25259/jrhm_17_2020 ◽

2021 ◽

Vol 2 ◽

pp. 4

Author(s):

Seema Dua ◽

Kamlesh Kumari Bajwa ◽

Atul Prashar ◽

Sonu Bansal ◽

Madhuri Beniwal ◽

...

Keyword(s):

Genome Editing ◽

Dna Sequences ◽

Nuclear Dna ◽

Genetic Material ◽

Underlying Mechanism ◽

Farm Animals ◽

Zinc Finger Nucleases ◽

Transcription Activator ◽

Minimal Impact ◽

Livestock Productivity

To cater the exponential growth of human population, need to improve food production and quality through modern biotechnology with limited recourses in a way that has minimal impact on the environment. The selective breeding and genomic selection have attended the momentum gain in livestock productivity. Recent advancement in genome-editing technologies offers exciting prospects for the production of healthy and prolific livestock. Genome editing involves altering genetic material by manipulation, addition, or removal of certain deoxyribonucleic acid (DNA) sequences at a specific locus in a way that does not occur naturally. The major genome editors are zinc finger nucleases, transcription-activator-like endonucleases, and clustered regularly interspaced short palindromic repeats associated protein nine systems which are proficient of cutting the nuclear DNA precisely at a predetermined position. This review provides an update on the use of genome editing systems to modify the genes related to reproduction of farm animal vis-à-vis human, update knowledge on the underlying mechanism and discusses new opportunities to produce genetically modified farm animals.

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CRISPR/Cas9 gene-editing strategies in cardiovascular cells

Cardiovascular Research ◽

10.1093/cvr/cvz250 ◽

2019 ◽

Vol 116 (5) ◽

pp. 894-907 ◽

Cited By ~ 1

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.

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