Targeted genome modification mediated by homology-directed repair and non-homologous end joining and its application

2019 ◽  
Vol 3 (3) ◽  
Keyword(s):  
End Joining ◽  
Homology Directed Repair ◽  
Genome Modification ◽  
Targeted Genome Modification ◽  
Non Homologous End Joining

scholarly journals Gene targeting facilitated by engineered sequence-specific nucleases and its potential for applications in crop improvement

2021 ◽  
Author(s):  
Daisuke Miki ◽  
Rui Wang ◽  
Jing Li ◽  
Dali Kong ◽  
Lei Zhang ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Agronomic Traits ◽  
Higher Plants ◽  
Crop Improvement ◽  
Strand Break ◽  
End Joining ◽  
Homology Directed Repair ◽  
Target Dna ◽  
Targeted Gene Editing ◽  
Non Homologous End Joining

Abstract Humans are currently facing the problem of how to ensure that there is enough food to feed all of the world’s population. Ensuring that the food supply is sufficient will likely require the modification of crop genomes to improve their agronomic traits. The development of engineered sequence-specific nucleases (SSNs) paved the way for targeted gene editing in organisms, including plants. SSNs generate a double-strand break (DSB) at the target DNA site in a sequence-specific manner. These DSBs are predominantly repaired via error-prone non-homologous end joining (NHEJ), and are only rarely repaired via error-free homology-directed repair (HDR) if an appropriate donor template is provided. Gene targeting (GT), i.e., the integration or replacement of a particular sequence, can be achieved with combinations of SSNs and repair donor templates. Although its efficiency is extremely low, GT has been achieved in some higher plants. Here, we provide an overview of SSN-facilitated GT in higher plants and discuss the potential of GT as a powerful tool for generating crop plants with desirable features.

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 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.

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 CopyCatchers are versatile active genetic elements that detect and quantify inter-homolog somatic gene conversion

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhiqian Li ◽  
Nimi Marcel ◽  
Sushil Devkota ◽  
Ankush Auradkar ◽  
Stephen M. Hedrick ◽  
...  
Keyword(s):  
Gene Conversion ◽  
Somatic Mutations ◽  
Genetic Screens ◽  
End Joining ◽  
Genetic Elements ◽  
Homology Directed Repair ◽  
Somatic Gene ◽  
Non Homologous End Joining ◽  
Or Gene ◽  
Therapy Strategies

AbstractCRISPR-based active genetic elements, or gene-drives, copied via homology-directed repair (HDR) in the germline, are transmitted to progeny at super-Mendelian frequencies. Active genetic elements also can generate widespread somatic mutations, but the genetic basis for such phenotypes remains uncertain. It is generally assumed that such somatic mutations are generated by non-homologous end-joining (NHEJ), the predominant double stranded break repair pathway active in somatic cells. Here, we develop CopyCatcher systems in Drosophila to detect and quantify somatic gene conversion (SGC) events. CopyCatchers inserted into two independent genetic loci reveal unexpectedly high rates of SGC in the Drosophila eye and thoracic epidermis. Focused RNAi-based genetic screens identify several unanticipated loci altering SGC efficiency, one of which (c-MYC), when downregulated, promotes SGC mediated by both plasmid and homologous chromosome-templates in human HEK293T cells. Collectively, these studies suggest that CopyCatchers can serve as effective discovery platforms to inform potential gene therapy strategies.

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 Efficient Disruption and Replacement of an Effector Gene in the Oomycete Phytophthora sojae using CRISPR/Cas9

2015 ◽  
Author(s):  
Yufeng Fang ◽  
Brett M Tyler
Keyword(s):  
Gene Replacement ◽  
Phytophthora Species ◽  
Phytophthora Sojae ◽  
Targeted Mutagenesis ◽  
End Joining ◽  
Soybean Seedlings ◽  
Homology Directed Repair ◽  
Effector Gene ◽  
Rxlr Effector ◽  
Non Homologous End Joining

Phytophthora sojae is a pathogenic oomycete that infects soybean seedlings as well as stems and roots of established plants, costing growers $1–2 billion per year. Due to its economic importance, P. sojae has become a model for the study of oomycete genetics, physiology and pathology. Despite the availability of several genome sequences, the lack of efficient techniques for targeted mutagenesis and gene replacement have long hampered genetic studies of pathogenicity in Phytophthora species. Here, we describe a CRISPR/Cas9 system enabling rapid and efficient genome editing in P. sojae. Using the RXLR effector gene Avr4/6 as target, we observed that in the absence of a homologous template, the repair of Cas9-induced double-strand breaks (DSBs) in P. sojae was mediated by non-homologous end joining (NHEJ), primarily resulting in short indels. Most mutants were homozygous, presumably due to gene conversion triggered by Cas9-mediated cleavage of non-mutant alleles. When donor DNA was present, homology directed repair (HDR) was observed, which resulted in the replacement of the target gene with the donor DNA. By testing the specific virulence of several NHEJ mutants and HDR -mediated gene replacements on soybeans, we have validated the contribution of Avr4/6 to recognition by soybean R gene loci, Rps4 and Rps6, but also uncovered additional contributions to resistance by these two loci. Our results establish a powerful tool for studying functional genomics in Phytophthora, which provides new avenues for better control of this pathogen.

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.

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