scholarly journals The Scope for Thalassemia Gene Therapy by Disruption of Aberrant Regulatory Elements

2019 ◽  
Vol 8 (11) ◽  
pp. 1959 ◽  
Author(s):  
Petros Patsali ◽  
Claudio Mussolino ◽  
Petros Ladas ◽  
Argyro Floga ◽  
Annita Kolnagou ◽  
...  
Keyword(s):  
High Efficiency ◽  
Regulatory Elements ◽  
Transcription Activator ◽  
End Joining ◽  
Advanced Therapy Medicinal Product ◽  
Systematic Analysis ◽  
Functional Correction ◽  
Advanced Therapy ◽  
Transgenic Cells ◽  
Non Homologous End Joining

The common IVSI-110 (G>A) β-thalassemia mutation is a paradigm for intronic disease-causing mutations and their functional repair by non-homologous end joining-mediated disruption. Such mutation-specific repair by disruption of aberrant regulatory elements (DARE) is highly efficient, but to date, no systematic analysis has been performed to evaluate disease-causing mutations as therapeutic targets. Here, DARE was performed in highly characterized erythroid IVSI-110(G>A) transgenic cells and the disruption events were compared with published observations in primary CD34+ cells. DARE achieved the functional correction of β-globin expression equally through the removal of causative mutations and through the removal of context sequences, with disruption events and the restriction of indel events close to the cut site closely resembling those seen in primary cells. Correlation of DNA-, RNA-, and protein-level findings then allowed the extrapolation of findings to other mutations by in silico analyses for potential repair based on the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9, Cas12a, and transcription activator-like effector nuclease (TALEN) platforms. The high efficiency of DARE and unexpected freedom of target design render the approach potentially suitable for 14 known thalassemia mutations besides IVSI-110(G>A) and put it forward for several prominent mutations causing other inherited diseases. The application of DARE, therefore, has a wide scope for sustainable personalized advanced therapy medicinal product development for thalassemia and beyond.

scholarly journals An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

2021 ◽  
Author(s):  
Eugene V. Gasanov ◽  
Justyna Jędrychowska ◽  
Michal Pastor ◽  
Malgorzata Wiweger ◽  
Axel Methner ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Target Site ◽  
Proof Of Concept ◽  
Intervention Site ◽  
End Joining ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Non Homologous End Joining

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

Editing livestock genomes with site-specific nucleases

2014 ◽  
Vol 26 (1) ◽  
pp. 74 ◽  
Author(s):  
Daniel F. Carlson ◽  
Wenfang Tan ◽  
Perry B. Hackett ◽  
Scott C. Fahrenkrug
Keyword(s):  
Genetic Alterations ◽  
Large Animal ◽  
Dna Breaks ◽  
Transcription Activator ◽  
End Joining ◽  
Site Specific ◽  
Homology Directed Repair ◽  
Double Strand Dna Breaks ◽  
Non Homologous End Joining ◽  
Inactive Genes

Over the past 5 years there has been a major transformation in our ability to precisely manipulate the genomes of animals. Efficiencies of introducing precise genetic alterations in large animal genomes have improved 100 000-fold due to a succession of site-specific nucleases that introduce double-strand DNA breaks with a specificity of 10–9. Herein we describe our applications of site-specific nucleases, especially transcription activator-like effector nucleases, to engineer specific alterations in the genomes of pigs and cows. We can introduce variable changes mediated by non-homologous end joining of DNA breaks to inactive genes. Alternatively, using homology-directed repair, we have introduced specific changes that support either precise alterations in a gene’s encoded polypeptide, elimination of the gene or replacement by another unrelated DNA sequence. Depending on the gene and the mutation, we can achieve 10%–50% effective rates of precise mutations. Applications of the new precision genetics are extensive. Livestock now can be engineered with selected phenotypes that will augment their value and adaption to variable ecosystems. In addition, animals can be engineered to specifically mimic human diseases and disorders, which will accelerate the production of reliable drugs and devices. Moreover, animals can be engineered to become better providers of biomaterials used in the medical treatment of diseases and disorders.

scholarly journals Drag-and-drop genome insertion without DNA cleavage with CRISPR-directed integrases

2021 ◽  
Author(s):  
Eleonora I. Ioannidi ◽  
Matthew T. N. Yarnall ◽  
Cian Schmitt-Ulms ◽  
Rohan N. Krajeski ◽  
Justin Lim ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
High Efficiency ◽  
End Joining ◽  
Differentiated Cells ◽  
Dividing Cells ◽  
Gene Integration ◽  
Fluorescent Tagging ◽  
Non Homologous End Joining ◽  
Drag And Drop

Programmable and multiplexed genome integration of large, diverse DNA cargo independent of DNA repair remains an unsolved challenge of genome editing. Current gene integration approaches require double-strand breaks that evoke DNA damage responses and rely on repair pathways that are inactive in terminally differentiated cells. Furthermore, CRISPR-based approaches that bypass double stranded breaks, such as Prime editing, are limited to modification or insertion of short sequences. We present Programmable Addition via Site-specific Targeting Elements, or PASTE, which achieves efficient and versatile gene integration at diverse loci by directing insertion with a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase. Without generating double stranded breaks, we demonstrate integration of sequences as large as ~36 kb with rates between 10-50% at multiple genomic loci across three human cell lines, primary T cells, and quiescent non-dividing primary human hepatocytes. To further improve PASTE, we discover thousands of novel serine integrases and cognate attachment sites from metagenomes and engineer active orthologs for high-efficiency integration using PASTE. We apply PASTE to fluorescent tagging of proteins, integration of therapeutically relevant genes, and production and secretion of transgenes. Leveraging the orthogonality of serine integrases, we engineer PASTE for multiplexed gene integration, simultaneously integrating three different genes at three genomic loci. PASTE has editing efficiencies comparable to or better than those of homology directed repair or non-homologous end joining based integration, with activity in non-dividing cells and fewer detectable off-target events. For therapeutic applications, PASTE can be delivered as mRNA with synthetically modified guides to programmably direct insertion of DNA templates carried by AAV or adenoviral vectors. PASTE expands the capabilities of genome editing via drag-and-drop gene integration, offering a platform with wide applicability for research, cell engineering, and gene therapy.

scholarly journals Effects of mitoTALENs-Directed Double-Strand Breaks on Plant Mitochondrial Genomes

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 153
Author(s):  
Shin-ichi Arimura
Keyword(s):  
Double Strand Breaks ◽  
Mitochondrial Genomes ◽  
Transcription Activator ◽  
End Joining ◽  
Strand Breaks ◽  
Large Deletions ◽  
Naturally Occurring ◽  
Copy Numbers ◽  
Nuclear Genomes ◽  
Non Homologous End Joining

Mitochondrial genomes in flowering plants differ from those in animals and yeasts in several ways, including having large and variable sizes, circular, linear and branched structures, long repeat sequences that participate in homologous recombinations, and variable genes orders, even within a species. Understanding these differences has been hampered by a lack of genetic methods for transforming plant mitochondrial genomes. We recently succeeded in disrupting targeted genes in mitochondrial genomes by mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs) in rice, rapeseed, and Arabidopsis. Double-strand breaks created by mitoTALENs were repaired not by non-homologous end-joining (NHEJ) but by homologous recombination (HR) between repeats near and far from the target sites, resulting in new genomic structures with large deletions and different configurations. On the other hand, in mammals, TALENs-induced DSBs cause small insertions or deletions in nuclear genomes and degradation of mitochondrial genomes. These results suggest that the mitochondrial and nuclear genomes of plants and mammals have distinct mechanisms for responding to naturally occurring DSBs. The different responses appear to be well suited to differences in size and copy numbers of each genome.

Genome Editing Tools und ihr Einsatz in der experimentellen Augenheilkunde

2017 ◽  
Vol 234 (03) ◽  
pp. 329-334
Author(s):  
M. Yanik ◽  
W. Wende ◽  
K. Stieger
Keyword(s):  
Genome Editing ◽  
Neurodegenerative Erkrankungen ◽  
Transcription Activator ◽  
End Joining ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

ZusammenfassungNeue molekularbiologische Werkzeuge revolutionieren zurzeit die Genomchirurgie (genome editing) mit weitreichendem Einfluss auch auf die experimentelle Augenheilkunde. Neben den bereits etablierten Systemen wie den Zinkfingernukleasen (ZFN) oder Transcription-activator-like-Effector-Nukleasen (TALEN) sind es insbesondere die CRISPR-/Cas-Systeme (CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR-associated), die überraschend einfach einen gezielten und präzisen Schnitt im Genom lebender Zellen ermöglichen. Dieser DNA-Doppelstrangbruch wird in der Zelle mittels NHEJ (non-homologous end joining) oder HDR (homology directed repair) repariert und kann ausgenutzt werden, um ein defektes Gen zu deaktivieren oder mithilfe einer korrekten Gensequenz zu reparieren. Die Genome-Editing-Technologie eröffnet damit bisher ungeahnte Möglichkeiten in der Grundlagenforschung, Biotechnologie, biomedizinischen Forschung bis hin zu ersten klinischen Anwendungen. Neurodegenerative Erkrankungen der Netzhaut stehen dabei aufgrund der guten Zugänglichkeit und des Immunprivilegs des Auges mit im Fokus des Interesses von Forschern und Firmen.

scholarly journals The Problem of the Low Rates of CRISPR/Cas9-Mediated Knock-ins in Plants: Approaches and Solutions

2019 ◽  
Vol 20 (13) ◽  
pp. 3371 ◽  
Author(s):  
Serge M. Rozov ◽  
Natalya V. Permyakova ◽  
Elena V. Deineko
Keyword(s):  
Genome Editing ◽  
Strand Break ◽  
Transcription Activator ◽  
End Joining ◽  
Knock Out ◽  
Genome Region ◽  
Higher Eukaryotes ◽  
Non Homologous End Joining ◽  
Recombination Pathway ◽  
Donor Template

The main number of genome editing events in plant objects obtained during the last decade with the help of specific nucleases zinc finger (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas are the microindels causing frameshift and subsequent gene knock-out. The knock-ins of genes or their parts, i.e., the insertion of them into a target genome region, are between one and two orders of magnitude less frequent. First and foremost, this is associated with the specific features of the repair systems of higher eukaryotes and the availability of the donor template in accessible proximity during double-strand break (DSB) repair. This review briefs the main repair pathways in plants according to the aspect of their involvement in genome editing. The main methods for increasing the frequency of knock-ins are summarized both along the homologous recombination pathway and non-homologous end joining, which can be used for plant objects.

scholarly journals Efficient Gene Disruption via Base Editing Induced Stop in Newt Pleurodeles waltl

Genes ◽  
2019 ◽  
Vol 10 (11) ◽  
pp. 837
Author(s):  
Cai ◽  
Peng ◽  
Ren ◽  
Wang
Keyword(s):  
High Efficiency ◽  
Loss Of Function ◽  
End Joining ◽  
Strand Breaks ◽  
Base Editing ◽  
Efficient Gene ◽  
Albino Phenotype ◽  
Non Homologous End Joining ◽  
Pleurodeles Waltl ◽  
Salamander Species

Loss-of-function approaches provide strong evidence for determining the role of particular genes. The prevalent CRISPR/Cas9 technique is widely used to disrupt target gene with uncontrolled non-homologous end joining after the double strand breaks, which results in mosaicism and multiple genotypes in the founders. In animal models with long generation time such as the salamanders, producing homozygous offspring mutants would be rather labor intensive and time consuming. Here we utilized the base editing technique to create the loss-of-function F0 mutants without the random indels. As a proof of principle, we successfully introduced premature stop codons into the tyrosinase locus and produced the albino phenotype in the newts (Pleurodeles waltl). We further demonstrated that the knockout efficiency could be greatly improved by using multiplex sgRNAs target the same gene. The F0 mutated animals showed fully loss-of-function by both genotyping and phenotyping analysis, which could enable direct functional analysis in the founders and avoid sophisticated breeding. This study not only presented the high efficiency of single base editing in a gigantic animal genome (>20 G), but also provided new tools for interrogating gene function in other salamander species.

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 Canonical DNA non-homologous end-joining; capacity versus fidelity

2020 ◽  
Vol 93 (1115) ◽  
pp. 20190966 ◽  
Author(s):  
Atsushi Shibata ◽  
Penny A Jeggo
Keyword(s):  
High Efficiency ◽  
High Capacity ◽  
Active Regions ◽  
Strand Break ◽  
Human Cells ◽  
End Joining ◽  
Dsb Repair ◽  
First Choice ◽  
Radiation Induced ◽  
Non Homologous End Joining

The significance of canonical DNA non-homologous end-joining (c-NHEJ) for DNA double strand break (DSB) repair has increased from lower organisms to higher eukaryotes, and plays the predominant role in human cells. Ku, the c-NHEJ end-binding component, binds DSBs with high efficiency enabling c-NHEJ to be the first choice DSB repair pathway, although alternative pathways can ensue after regulated steps to remove Ku. Indeed, radiation-induced DSBs are repaired rapidly in human cells. However, an important question is the fidelity with which radiation-induced DSBs are repaired, which is essential for assessing any harmful impacts caused by radiation exposure. Indeed, is compromised fidelity a price we pay for high capacity repair. Two subpathways of c-NHEJ have been revealed; a fast process that does not require nucleases or significant chromatin changes and a slower process that necessitates resection factors, and potentially more significant chromatin changes at the DSB. Recent studies have also shown that DSBs within transcriptionally active regions are repaired by specialised mechanisms, and the response at such DSBs encompasses a process of transcriptional arrest. Here, we consider the limitations of c-NHEJ that might result in DSB misrepair. We consider the common IR-induced misrepair events and discuss how they might arise via the distinct subpathways of c-NHEJ.

scholarly journals High Homology-Directed Repair Using Mitosis Phase and Nucleus Localizing Signal

2020 ◽  
Vol 21 (11) ◽  
pp. 3747
Author(s):  
Jeong Pil Han ◽  
Yoo Jin Chang ◽  
Dong Woo Song ◽  
Beom Seok Choi ◽  
Ok Jae Koo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
High Efficiency ◽  
End Joining ◽  
Homology Directed Repair ◽  
Cas9 Protein ◽  
Localization Signal ◽  
Non Homologous End Joining ◽  
The Relationship ◽  
Donor Template

In homology-directed repair, mediated knock-in single-stranded oligodeoxynucleotides (ssODNs) can be used as a homologous template and present high efficiency, but there is still a need to improve efficiency. Previous studies have mainly focused on controlling double-stranded break size, ssODN stability, and the DNA repair cycle. Nevertheless, there is a lack of research on the correlation between the cell cycle and single-strand template repair (SSTR) efficiency. Here, we investigated the relationship between cell cycle and SSTR efficiency. We found higher SSTR efficiency during mitosis, especially in the metaphase and anaphase. A Cas9 protein with a nuclear localization signal (NLS) readily migrated to the nucleus; however, the nuclear envelope inhibited the nuclear import of many nucleotide templates. This seemed to result in non-homologous end joining (NHEJ) before the arrival of the homologous template. Thus, we assessed whether NLS-tagged ssODNs and free NLS peptides could circumvent problems posed by the nuclear envelope. NLS-tagging ssODNs enhanced SSTR and indel efficiency by 4-fold compared to the control. Our results suggest the following: (1) mitosis is the optimal phase for SSTR, (2) the donor template needs to be delivered to the nucleus before nuclease delivery, and (3) NLS-tagging ssODNs improve SSTR efficiency, especially high in mitosis.

Sign in / Sign up

Export Citation Format

Share Document