scholarly journals Programmed genome editing of the omega-1 ribonuclease 1 of the blood fluke,Schistosoma mansoni

2018 ◽  
Author(s):  
Wannaporn Ittiprasert ◽  
Victoria H. Mann ◽  
Shannon E. Karinshak ◽  
Avril Coghlan ◽  
Gabriel Rinaldi ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Genome Editing ◽  
Human Macrophage ◽  
Wild Type ◽  
End Joining ◽  
Chromosomal Breakage ◽  
Knock Out ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

AbstractCRISPR/Cas9 based genome editing has yet been reported in parasitic or indeed any species of the phylum Platyhelminthes. We tested this approach by targeting omega-1 (ω1) ofSchistosoma mansonias a proof of principle. This secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative immuno-pathological readouts for programmed genome editing. Schistosome eggs were either exposed to Cas9 complexed with a synthetic guide RNA (sgRNA) complementary to exon 6 of ω1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor transgene that encoded six stop codons, flanked by 50 nt-long 5’-and 3’-microhomology arms matching the predicted Cas9-catalyzed double stranded break (DSB) within ω1. CRISPResso analysis of amplicons spanning the DSB revealed ∼4.5% of the reads were mutated by insertions, deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion of the donor transgene. Transcripts encoding ω1 were reduced >80% and lysates of ω1-edited eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the ω1 gene. Whereas lysates of wild type eggs polarized Th2 cytokine responses including IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of the cytokines followed the exposure to lysates of ω1-mutated schistosome eggs. Following injection of schistosome eggs into the tail vein of mice, the volume of pulmonary granulomas surrounding ω1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by homology directed repair and/or non-homologous end joining, and mutation of ω1 impeded the capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of pulmonary circumoval granulomas. Knock-out of ω1 and the impaired immunological phenotype showcase the novel application of programmed gene editing in and functional genomics for schistosomes.

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 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.

scholarly journals Genome-scale CRISPR screens are efficient in non-homologous end-joining deficient cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Joana Ferreira da Silva ◽  
Sejla Salic ◽  
Marc Wiedner ◽  
Paul Datlinger ◽  
Patrick Essletzbichler ◽  
...  
Keyword(s):  
Genome Editing ◽  
Large Scale ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Knock Out ◽  
Genetic Ablation ◽  
Alternative End Joining ◽  
Non Homologous End Joining ◽  
Genome Scale

Abstract The mutagenic repair of Cas9 generated breaks is thought to predominantly rely on non-homologous end-joining (NHEJ), leading to insertions and deletions within DNA that culminate in gene knock-out (KO). In this study, by taking focused as well as genome-wide approaches, we show that this pathway is dispensable for the repair of such lesions. Genetic ablation of NHEJ is fully compensated for by alternative end joining (alt-EJ), in a POLQ-dependent manner, resulting in a distinct repair signature with larger deletions that may be exploited for large-scale genome editing. Moreover, we show that cells deficient for both NHEJ and alt-EJ were still able to repair CRISPR-mediated DNA double-strand breaks, highlighting how little is yet known about the mechanisms of CRISPR-based genome editing.

scholarly journals Simultaneous precise editing of multiple genes in human cells

2019 ◽  
Vol 47 (19) ◽  
pp. e116-e116 ◽  
Author(s):  
Stephan Riesenberg ◽  
Manjusha Chintalapati ◽  
Dominik Macak ◽  
Philipp Kanis ◽  
Tomislav Maricic ◽  
...  
Keyword(s):  
Genome Editing ◽  
Kinase Inhibitor ◽  
Double Strand Breaks ◽  
End Joining ◽  
Nucleotide Substitutions ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
A Genome ◽  
Dependent Protein ◽  
Non Homologous End Joining

Abstract When double-strand breaks are introduced in a genome by CRISPR they are repaired either by non-homologous end joining (NHEJ), which often results in insertions or deletions (indels), or by homology-directed repair (HDR), which allows precise nucleotide substitutions to be introduced if a donor oligonucleotide is provided. Because NHEJ is more efficient than HDR, the frequency with which precise genome editing can be achieved is so low that simultaneous editing of more than one gene has hitherto not been possible. Here, we introduced a mutation in the human PRKDC gene that eliminates the kinase activity of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This results in an increase in HDR irrespective of cell type and CRISPR enzyme used, sometimes allowing 87% of chromosomes in a population of cells to be precisely edited. It also allows for precise editing of up to four genes simultaneously (8 chromosomes) in the same cell. Transient inhibition of DNA-PKcs by the kinase inhibitor M3814 is similarly able to enhance precise genome editing.

scholarly journals Precision genome editing in the CRISPR era

2017 ◽  
Vol 95 (2) ◽  
pp. 187-201 ◽  
Author(s):  
Jayme Salsman ◽  
Graham Dellaire
Keyword(s):  
Gene Therapy ◽  
Genome Editing ◽  
Point Mutations ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Genetic Changes ◽  
New Era ◽  
Homology Directed Repair ◽  
Repair Template ◽  
Non Homologous End Joining

With the introduction of precision genome editing using CRISPR–Cas9 technology, we have entered a new era of genetic engineering and gene therapy. With RNA-guided endonucleases, such as Cas9, it is possible to engineer DNA double strand breaks (DSB) at specific genomic loci. DSB repair by the error-prone non-homologous end-joining (NHEJ) pathway can disrupt a target gene by generating insertions and deletions. Alternatively, Cas9-mediated DSBs can be repaired by homology-directed repair (HDR) using an homologous DNA repair template, thus allowing precise gene editing by incorporating genetic changes into the repair template. HDR can introduce gene sequences for protein epitope tags, delete genes, make point mutations, or alter enhancer and promoter activities. In anticipation of adapting this technology for gene therapy in human somatic cells, much focus has been placed on increasing the fidelity of CRISPR–Cas9 and increasing HDR efficiency to improve precision genome editing. In this review, we will discuss applications of CRISPR technology for gene inactivation and genome editing with a focus on approaches to enhancing CRISPR–Cas9-mediated HDR for the generation of cell and animal models, and conclude with a discussion of recent advances and challenges towards the application of this technology for gene therapy in humans.

scholarly journals Pervasive head-to-tail insertions of DNA templates mask desired CRISPR/Cas9-mediated genome editing events

2019 ◽  
Author(s):  
Boris V. Skryabin ◽  
Leonid Gubar ◽  
Birte Seeger ◽  
Helena Kaiser ◽  
Anja Stegemann ◽  
...  
Keyword(s):  
High Rate ◽  
Model Organisms ◽  
Pcr Analysis ◽  
End Joining ◽  
Dna Templates ◽  
Knock Out ◽  
Homology Directed Repair ◽  
Wide Range ◽  
Non Homologous End Joining ◽  
Knock Out Mouse

AbstractCRISPR/Cas9 mediated homology-directed DNA repair is the method of choice for precise gene editing in a wide range of model organisms, including mouse and human. Broad use by the biomedical community refined the method, making it more efficient and sequence specific. Nevertheless, the rapidly evolving technique still contains pitfalls. During the generation of six different conditional knock-out mouse models, we discovered that frequently (sometimes solely) homology-directed repair and/or non-homologous end-joining mechanisms caused multiple unwanted head-to-tail insertions of donor DNA templates. Disturbingly, conventionally applied PCR analysis—in most cases—failed to identify such multiple integration events, which led to a high rate of falsely claimed precisely edited alleles. We caution that comprehensive analysis of modified alleles is essential, and offer practical solutions to correctly identify precisely edited chromosomes.

scholarly journals Efficient single-copy HDR by 5’ modified long dsDNA donors

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Jose Arturo Gutierrez-Triana ◽  
Tinatini Tavhelidse ◽  
Thomas Thumberger ◽  
Isabelle Thomas ◽  
Beate Wittbrodt ◽  
...  
Keyword(s):  
Genome Editing ◽  
Single Copy ◽  
Gene Replacement ◽  
End Joining ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

CRISPR/Cas9 efficiently induces targeted mutations via non-homologous-end-joining but for genome editing, precise, homology-directed repair (HDR) of endogenous DNA stretches is a prerequisite. To favor HDR, many approaches interfere with the repair machinery or manipulate Cas9 itself. Using Medaka we show that the modification of 5’ ends of long dsDNA donors strongly enhances HDR, favors efficient single-copy integration by retaining a monomeric donor conformation thus facilitating successful gene replacement or tagging.

scholarly journals oca2 targeting using CRISPR/Cas9 in the Malawi cichlid Astatotilapia calliptera

2022 ◽  
Author(s):  
Bethan Clark ◽  
Joel Elkin ◽  
Aleksandra Marconi ◽  
George F Turner ◽  
Alan M Smith ◽  
...  
Keyword(s):  
Lake Malawi ◽  
Large Deletion ◽  
Colour Pattern ◽  
Coding Region ◽  
Genetic Loci ◽  
End Joining ◽  
Knock Out ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

Identifying genetic loci underlying trait variation provides insights into the mechanisms of diversification, but demonstrating causality and characterising the role of genetic loci requires testing candidate gene function, often in non-model species. Here we establish CRISPR/Cas9 editing in Astatotilapia calliptera, a generalist cichlid of the remarkably diverse Lake Malawi radiation. By targeting the gene oca2 required for melanin synthesis in other vertebrate species, we show efficient editing and germline transmission. Gene edits include indels in the coding region, likely a result of non-homologous end joining, and a large deletion in the 3′ UTR due to homology-directed repair. We find that oca2 knock-out A. calliptera lack melanin, which may be useful for developmental imaging in embryos and studying colour pattern formation in adults. As A. calliptera resembles the presumed generalist ancestor of the Lake Malawi cichlids radiation, establishing genome editing in this species will facilitate investigating speciation, adaptation and trait diversification in this textbook radiation.

scholarly journals Approaches to Enhance Precise CRISPR/Cas9-Mediated Genome Editing

2021 ◽  
Vol 22 (16) ◽  
pp. 8571
Author(s):  
Christopher E. Denes ◽  
Alexander J. Cole ◽  
Yagiz Alp Aksoy ◽  
Geng Li ◽  
G. Gregory Neely ◽  
...  
Keyword(s):  
Dna Repair ◽  
Genome Editing ◽  
Nuclear Dna ◽  
Great Promise ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Repair Pathways ◽  
Non Homologous End Joining ◽  
Small Molecule Modulators

Modification of the human genome has immense potential for preventing or treating disease. Modern genome editing techniques based on CRISPR/Cas9 show great promise for altering disease-relevant genes. The efficacy of precision editing at CRISPR/Cas9-induced double-strand breaks is dependent on the relative activities of nuclear DNA repair pathways, including the homology-directed repair and error-prone non-homologous end-joining pathways. The competition between multiple DNA repair pathways generates mosaic and/or therapeutically undesirable editing outcomes. Importantly, genetic models have validated key DNA repair pathways as druggable targets for increasing editing efficacy. In this review, we highlight approaches that can be used to achieve the desired genome modification, including the latest progress using small molecule modulators and engineered CRISPR/Cas proteins to enhance precision editing.

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.

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