scholarly journals CRISPR FokI Dead Cas9 System: Principles and Applications in Genome Engineering

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2518
Author(s):  
Maryam Saifaldeen ◽  
Dana E. Al-Ansari ◽  
Dindial Ramotar ◽  
Mustapha Aouida
Keyword(s):  
Gene Editing ◽  
Genome Engineering ◽  
Catalytic Domain ◽  
Zinc Finger Nucleases ◽  
Dna Double Strand Breaks ◽  
Transcription Activator ◽  
Strand Breaks ◽  
Protospacer Adjacent Motif ◽  
Cas9 Protein ◽  
Wide Range

The identification of the robust clustered regularly interspersed short palindromic repeats (CRISPR) associated endonuclease (Cas9) system gene-editing tool has opened up a wide range of potential therapeutic applications that were restricted by more complex tools, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Nevertheless, the high frequency of CRISPR system off-target activity still limits its applications, and, thus, advanced strategies for highly specific CRISPR/Cas9-mediated genome editing are continuously under development including CRISPR–FokI dead Cas9 (fdCas9). fdCas9 system is derived from linking a FokI endonuclease catalytic domain to an inactive Cas9 protein and requires a pair of guide sgRNAs that bind to the sense and antisense strands of the DNA in a protospacer adjacent motif (PAM)-out orientation, with a defined spacer sequence range around the target site. The dimerization of FokI domains generates DNA double-strand breaks, which activates the DNA repair machinery and results in genomic edit. So far, all the engineered fdCas9 variants have shown promising gene-editing activities in human cells when compared to other platforms. Herein, we review the advantages of all published variants of fdCas9 and their current applications in genome engineering.

scholarly journals Various Aspects of a Gene Editing System—CRISPR–Cas9

2020 ◽  
Vol 21 (24) ◽  
pp. 9604
Author(s):  
Edyta Janik ◽  
Marcin Niemcewicz ◽  
Michal Ceremuga ◽  
Lukasz Krzowski ◽  
Joanna Saluk-Bijak ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Genome Engineering ◽  
High Efficiency ◽  
Adaptive Immune System ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Guide Rna ◽  
Adaptive Immune ◽  
Cas9 Protein

The discovery of clustered, regularly interspaced short palindromic repeats (CRISPR) and their cooperation with CRISPR-associated (Cas) genes is one of the greatest advances of the century and has marked their application as a powerful genome engineering tool. The CRISPR–Cas system was discovered as a part of the adaptive immune system in bacteria and archaea to defend from plasmids and phages. CRISPR has been found to be an advanced alternative to zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) for gene editing and regulation, as the CRISPR–Cas9 protein remains the same for various gene targets and just a short guide RNA sequence needs to be altered to redirect the site-specific cleavage. Due to its high efficiency and precision, the Cas9 protein derived from the type II CRISPR system has been found to have applications in many fields of science. Although CRISPR–Cas9 allows easy genome editing and has a number of benefits, we should not ignore the important ethical and biosafety issues. Moreover, any tool that has great potential and offers significant capabilities carries a level of risk of being used for non-legal purposes. In this review, we present a brief history and mechanism of the CRISPR–Cas9 system. We also describe on the applications of this technology in gene regulation and genome editing; the treatment of cancer and other diseases; and limitations and concerns of the use of CRISPR–Cas9.

scholarly journals Genome editing for blood disorders: state of the art and recent advances

2019 ◽  
Vol 3 (3) ◽  
pp. 289-299 ◽  
Author(s):  
Marianna Romito ◽  
Rajeev Rai ◽  
Adrian J. Thrasher ◽  
Alessia Cavazza
Keyword(s):  
Gene Editing ◽  
Therapeutic Potential ◽  
State Of The Art ◽  
Clinical Success ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Blood Disorders ◽  
Flexible Tool ◽  
Wide Range ◽  
Made In

Abstract In recent years, tremendous advances have been made in the use of gene editing to precisely engineer the genome. This technology relies on the activity of a wide range of nuclease platforms — such as zinc-finger nucleases, transcription activator-like effector nucleases, and the CRISPR–Cas system — that can cleave and repair specific DNA regions, providing a unique and flexible tool to study gene function and correct disease-causing mutations. Preclinical studies using gene editing to tackle genetic and infectious diseases have highlighted the therapeutic potential of this technology. This review summarizes the progresses made towards the development of gene editing tools for the treatment of haematological disorders and the hurdles that need to be overcome to achieve clinical success.

scholarly journals Expanding the CRISPR Toolbox with ErCas12a in Zebrafish and Human Cells

2019 ◽  
Author(s):  
Wesley A. Wierson ◽  
Brandon W. Simone ◽  
Zachary WareJoncas ◽  
Carla Mann ◽  
Jordan M. Welker ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Human Cells ◽  
Effector Proteins ◽  
Dna Double Strand Breaks ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
Strand Breaks ◽  
Guide Rnas ◽  
Protospacer Adjacent Motif ◽  
Strand Annealing

AbstractClustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) effector proteins enable the targeting of DNA double-strand breaks (DSBs) to defined loci based on a variable length RNA guide specific to each effector. The guide RNAs are generally similar in size and form, consisting of a ~20 nucleotide sequence complementary to the DNA target and an RNA secondary structure recognized by the effector. However, the effector proteins vary in Protospacer Adjacent Motif (PAM) requirements, nuclease activities, and DNA binding kinetics. Recently, ErCas12a, a new member of the Cas12a family, was identified in Eubacterium rectale. Here, we report the first characterization of ErCas12a activity in zebrafish and human cells. Using a fluorescent reporter system, we show that CRISPR/ErCas12a elicits strand annealing mediated DNA repair more efficiently than CRISPR/Cas9. Further, using our previously reported gene targeting method that utilizes short homology, GeneWeld, we demonstrate the use of CRISPR/ErCas12a to integrate reporter alleles into the genomes of both zebrafish and human cells. Together, this work provides methods for deploying an additional CRISPR/Cas system, thus increasing the flexibility researchers have in applying genome engineering technologies.

scholarly journals Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

Biology ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 530
Author(s):  
Marlo K. Thompson ◽  
Robert W. Sobol ◽  
Aishwarya Prakash
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Gene Editing ◽  
Adaptive Immune System ◽  
Zinc Finger Nucleases ◽  
Specific Gene ◽  
Target Sequence ◽  
Transcription Activator ◽  
Low Cytotoxicity

The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

scholarly journals Mini-Review Regarding the Applicability of Genome Editing Techniques Developed for Studying Infertility

Diagnostics ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 246
Author(s):  
Bogdan Doroftei ◽  
Ovidiu-Dumitru Ilie ◽  
Maria Puiu ◽  
Alin Ciobica ◽  
Ciprian Ilea
Keyword(s):  
Motor Activity ◽  
Experimental Model ◽  
Zinc Finger ◽  
Biomedical Research ◽  
Zinc Finger Nucleases ◽  
Loss Of Function ◽  
Transcription Activator ◽  
Phenotypic Changes ◽  
Wide Range ◽  
Short Palindromic Repeat

Infertility is a highly debated topic today. It has been long hypothesized that infertility has an idiopathic cause, but recent studies demonstrated the existence of a genetic substrate. Fortunately, the methods of editing the human genome proven to be revolutionary. Following research conducted, we identified a total of 21 relevant studies; 14 were performed on mice, 5 on zebrafish and 2 on rats. We concluded that over forty-four genes in total are dispensable for fertility in both sexes without affecting host homeostasis. However, there are genes whose loss-of-function induces moderate to severe phenotypic changes in both sexes. There were situations in which the authors reported infertility, exhibited by the experimental model, or other pathologies such as cryptorchidism, cataracts, or reduced motor activity. Overall, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 are techniques that offer a wide range of possibilities for studying infertility, even to create mutant variants. It can be concluded that ZFNs, TALENs, and CRISPR/Cas9 are crucial tools in biomedical research.

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

Acta Naturae ◽  
2014 ◽  
Vol 6 (3) ◽  
pp. 19-40 ◽  
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.

Gene editing as a promising approach for respiratory diseases

2018 ◽  
Vol 55 (3) ◽  
pp. 143-149 ◽  
Author(s):  
Yichun Bai ◽  
Yang Liu ◽  
Zhenlei Su ◽  
Yana Ma ◽  
Chonghua Ren ◽  
...  
Keyword(s):  
Respiratory Diseases ◽  
Gene Editing ◽  
Gene Mutations ◽  
Well Being ◽  
Short Review ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Mortality And Morbidity ◽  
Chronic Respiratory Diseases ◽  
Difficulty Breathing

Respiratory diseases, which are leading causes of mortality and morbidity in the world, are dysfunctions of the nasopharynx, the trachea, the bronchus, the lung and the pleural cavity. Symptoms of chronic respiratory diseases, such as cough, sneezing and difficulty breathing, may seriously affect the productivity, sleep quality and physical and mental well-being of patients, and patients with acute respiratory diseases may have difficulty breathing, anoxia and even life-threatening respiratory failure. Respiratory diseases are generally heterogeneous, with multifaceted causes including smoking, ageing, air pollution, infection and gene mutations. Clinically, a single pulmonary disease can exhibit more than one phenotype or coexist with multiple organ disorders. To correct abnormal function or repair injured respiratory tissues, one of the most promising techniques is to correct mutated genes by gene editing, as some gene mutations have been clearly demonstrated to be associated with genetic or heterogeneous respiratory diseases. Zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) systems are three innovative gene editing technologies developed recently. In this short review, we have summarised the structure and operating principles of the ZFNs, TALENs and CRISPR/Cas9 systems and their preclinical and clinical applications in respiratory diseases.

scholarly journals The Role of Gene Editing in Neurodegenerative Diseases

2018 ◽  
Vol 27 (3) ◽  
pp. 364-378 ◽  
Author(s):  
Hueng-Chuen Fan ◽  
Ching-Shiang Chi ◽  
Yih-Jing Lee ◽  
Jeng-Dau Tsai ◽  
Shinn-Zong Lin ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Gene Editing ◽  
Clinical Manifestations ◽  
Zinc Finger Nucleases ◽  
Diagnostic Tools ◽  
Pathological Feature ◽  
Transcription Activator ◽  
Substantial Impact ◽  
Parkinson’S Diseases ◽  
The Impact

Neurodegenerative diseases (NDs), at least including Alzheimer’s, Huntington’s, and Parkinson’s diseases, have become the most dreaded maladies because there are no precise diagnostic tools or definite treatments for these debilitating diseases. The increased prevalence and a substantial impact on the social–economic and medical care of NDs propel governments to develop policies to counteract the impact. Although the etiologies of NDs are still unknown, growing evidence suggests that genetic, cellular, and circuit alternations may cause the generation of abnormal misfolded proteins, which uncontrolledly accumulate to damage and eventually overwhelm the protein-disposal mechanisms of these neurons, leading to a common pathological feature of NDs. If the functions and the connectivity can be restored, alterations and accumulated damages may improve. The gene-editing tools including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats–associated nucleases (CRISPR/CAS) have emerged as a novel tool not only for generating specific ND animal models for interrogating the mechanisms and screening potential drugs against NDs but also for the editing sequence-specific genes to help patients with NDs to regain function and connectivity. This review introduces the clinical manifestations of three distinct NDs and the applications of the gene-editing technology on these debilitating diseases.

Genome Editing Toolbox

Blood ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document