scholarly journals A CyclinB2-Cas9 fusion promotes the homology-directed repair of double-strand breaks

2019 ◽  
Author(s):  
Manuel M. Vicente ◽  
Afonso Mendes ◽  
Margarida Cruz ◽  
José R. Vicente ◽  
Vasco M. Barreto
Keyword(s):  
Cell Cycle ◽  
Strand Break ◽  
Relative Increase ◽  
End Joining ◽  
New Era ◽  
Homology Directed Repair ◽  
Guide Rnas ◽  
Dna Lesion ◽  
Non Homologous End Joining ◽  
Cycle Dependent

ABSTRACTThe discovery of clustered regularly interspaced palindromic repeats (CRISPR), a defense system against viruses found in bacteria, launched a new era in gene targeting. The key feature of this technique is the guiding of the endonuclease Cas9 by single guide RNAs (sgRNA) to specific sequences, where a DNA lesion is introduced to trigger DNA repair. The CRISPR/Cas9 system may be extremely relevant for gene therapy, but the technique needs improvement to become a safe and fully effective tool. The Cas9-induced double-strand break (DSB) is repaired by one of two pathways, the error-prone Non-homologous end joining (NHEJ) or the high-fidelity Homology Direct Repair (HDR). Shifting the repair of the DSB to HDR is challenging, given the efficiency of NHEJ. Here we describe an engineered protein approach to increase knock-in efficiency by promoting the relative increase in Cas9 activity in G2, the phase of the cell cycle where HDR is more active. Cas9 was fused to the degradation domain of proteins known to be degraded in G1. The activity of two chimeric proteins, Geminin-Cas9 and CyclinB2-Cas9, is demonstrated, as well as their cell-cycle-dependent degradation. The chimeras shifted the repair of the DSBs to the HDR repair pathway compared to the commonly used Cas9. The application of cell cycle specific degradation tags could pave the way for more efficient and secure gene editing applications of the CRISPR/Cas9 system.

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.

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 A Homology Independent Sequence Replacement Strategy in Human Cells Using a CRISPR Nuclease

2020 ◽  
Author(s):  
Eric Danner ◽  
Mikhail Lebedin ◽  
Kathrin de la Rosa ◽  
Ralf Kühn
Keyword(s):  
Basic Research ◽  
Strand Break ◽  
End Joining ◽  
Linear Amplification ◽  
Promising Alternative ◽  
Independent Sequence ◽  
Replacement Strategy ◽  
Homology Directed Repair ◽  
Dividing Cells ◽  
Non Homologous End Joining

AbstractPrecision genomic alterations largely rely on Homology Directed Repair (HDR), but targeting without homology using the Non-Homologous End Joining (NHEJ) pathway has gained attention as a promising alternative. Previous studies demonstrated precise insertions formed by the ligation of donor DNA into a targeted genomic double strand break in both dividing and non-dividing cells. Here we extend this idea and use NHEJ repair to replace genomic segments with donor sequences; we name this method ‘Replace’ editing (Rational end-joining protocol delivering a targeted sequence exchange). Using CRISPR/Cas9 we create two genomic breaks and ligate a donor sequence in-between. This exchange of a genomic for a donor sequence uses neither microhomology nor homology arms. We target four loci and show successful exchange of exons in 16% to 54% of cells. Using linear amplification methods and deep sequencing pipelines we quantify the diversity of outcomes following Replace editing and profile mutations formed at the ligated interfaces. The ability to replace exons or other genomic sequences in cells not efficiently modified by HDR holds promise for both basic research and medicine.

scholarly journals Multiple metabolic signals including AMPK and PKA regulate glucose-stimulated double strand break resection in yeast

2021 ◽  
Author(s):  
Stephanie Lomonaco ◽  
Dominic Bazzano ◽  
Thomas E Wilson
Keyword(s):  
Protein Kinase ◽  
Carbon Source ◽  
Late Phase ◽  
Strand Break ◽  
Primary Role ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Checkpoint Response ◽  
Non Homologous End Joining ◽  
Cycle Dependent

DNA double strand breaks (DSBs) are cytotoxic lesions repaired by non-homologous end joining (NHEJ) and homologous recombination (HR), with 5' strand resection being the committed step in transition from NHEJ to HR. We previously discovered that gal1 yeast, which cannot metabolize galactose, were unable to perform efficient 5' resection even though DSBs were formed. Adding glucose or restoring GAL1 restored resection, suggesting that carbon source metabolism signals to DSB repair. Here we demonstrate that any fermentable carbon source, including raffinose, can stimulate resection and that the stimulatory effect of glucose was associated with decreased, not increased, cellular ATP. The effect was cell cycle dependent and did not occur in G1, while glucose augmented the G2/M checkpoint arrest even in cells deficient in resection. AMP-activated protein kinase pathway mutants showed only low basal resection despite glucose addition but had normal checkpoint arrest, indicating a primary role for Snf1 specifically in glucose-stimulated resection. The metabolic inputs to resection were multifactorial, however, with loss of the transcriptional repressor Mig1 leading to increased basal resection, three distinct patterns of deficiency with loss of the protein kinase A catalytic subunits, Tpk1, Tpk2 andTpk3, and a resection delay in yeast lacking the lysine demethylase Rph1 that helped separate early and late phase responses to glucose. These results reveal multiple interrelated metabolic signals that optimize DSB resection efficiency while independently amplifying the G2/M checkpoint response.

scholarly journals Beta human papillomavirus 8 E6 allows colocalization of non-homologous end joining and homologous recombination repair factors.

2021 ◽  
Author(s):  
Nicholas Wallace ◽  
Changkun Hu ◽  
Taylor Bugbee ◽  
Rachel Palinski
Keyword(s):  
Cell Cycle ◽  
Human Papillomavirus ◽  
Homologous Recombination ◽  
Immunofluorescence Microscopy ◽  
Cell Cycle Distribution ◽  
End Joining ◽  
Dsb Repair ◽  
Dna Lesion ◽  
Non Homologous End Joining ◽  
Mutagenic Process

Beta human papillomavirus (β-HPV) are hypothesized to make DNA damage more mutagenic and potentially more carcinogenic. Double strand breaks in DNA (DSBs) are the most deleterious DNA lesion. They are typically repaired by homologous recombination (HR) or non-homologous end joining (NHEJ). HR occurs after DNA replication while NHEJ can occur at any point in the cell cycle. They are not thought to occur in the same cell at the same time. By destabilizing p300, β-HPV type 8 protein E6 (β-HPV8 E6) attenuates both repair pathways. However, β-HPV8 E6 delays rather than abrogates DSB repair. Thus, β-HPV8 E6 may cause DSBs to be repaired through a more mutagenic process. To evaluate this, immunofluorescence microscopy was used to detect colocalization, formation, and resolution of DSB repair complexes following damage. Flow cytometry and immunofluorescence microscopy were used to determine the cell cycle distribution of repair complexes. The resulting data show that β-HPV8 E6 causes HR factors (RPA70 and RAD51) to colocalize with a persistent NHEJ factor (pDNA-PKcs). RPA70 complexes gave way to RAD51 complexes as in canonical HR, but RAD51 and pDNA-PKcs colocalization did not resolve within 32 hours of damage. The persistent RAD51 foci occur in G1 phase, consistent with recruitment after NHEJ fails. Chemical inhibition of p300, p300 knockout cells, and an β-HPV8 E6 mutant demonstrate that these phenotypes are the result of β-HPV8 E6-meidated p300 destabilization. Mutations associated with DSB repair were identified using next generation sequencing after a CAS9-induced DSB. β-HPV8 E6 increases the frequency of mutations (>15 fold) and deletions (>20 fold) associated with DSB repair. These data suggest that β-HPV8 E6 causes abnormal DSB repair where both NHEJ and HR occur at the same lesion and that his leads to deletions as the single stranded DNA produced during HR is removed by NHEJ.

scholarly journals Concurrent genome and epigenome editing by CRISPR-mediated sequence replacement

BMC Biology ◽  
2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Jes Alexander ◽  
Gregory M. Findlay ◽  
Martin Kircher ◽  
Jay Shendure
Keyword(s):  
Genome Editing ◽  
Cpg Island ◽  
Strand Break ◽  
End Joining ◽  
Exogenous Dna ◽  
Cpg Dinucleotides ◽  
Epigenome Editing ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

Abstract Background Recent advances in genome editing have facilitated the direct manipulation of not only the genome, but also the epigenome. Genome editing is typically performed by introducing a single CRISPR/Cas9-mediated double-strand break (DSB), followed by non-homologous end joining (NHEJ)- or homology-directed repair-mediated repair. Epigenome editing, and in particular methylation of CpG dinucleotides, can be performed using catalytically inactive Cas9 (dCas9) fused to a methyltransferase domain. However, for investigations of the role of methylation in gene silencing, studies based on dCas9-methyltransferase have limited resolution and are potentially confounded by the effects of binding of the fusion protein. As an alternative strategy for epigenome editing, we tested CRISPR/Cas9 dual cutting of the genome in the presence of in vitro methylated exogenous DNA, with the aim of driving replacement of the DNA sequence intervening the dual cuts via NHEJ. Results In a proof of concept at the HPRT1 promoter, successful replacement events with heavily methylated alleles of a CpG island resulted in functional silencing of the HPRT1 gene. Although still limited in efficiency, our study demonstrates concurrent epigenome and genome editing in a single event. Conclusions This study opens the door to investigations of the functional consequences of methylation patterns at single CpG dinucleotide resolution. Our results furthermore support the conclusion that promoter methylation is sufficient to functionally silence gene expression.

scholarly journals The (Lack of) DNA Double-Strand Break Repair Pathway Choice During V(D)J Recombination

2022 ◽  
Vol 12 ◽  
Author(s):  
Alice Libri ◽  
Timea Marton ◽  
Ludovic Deriano
Keyword(s):  
Dna Repair ◽  
Gene Diversity ◽  
Receptor Gene ◽  
Strand Break ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Homology Directed Repair ◽  
Pathway Choice ◽  
Non Homologous End Joining

DNA double-strand breaks (DSBs) are highly toxic lesions that can be mended via several DNA repair pathways. Multiple factors can influence the choice and the restrictiveness of repair towards a given pathway in order to warrant the maintenance of genome integrity. During V(D)J recombination, RAG-induced DSBs are (almost) exclusively repaired by the non-homologous end-joining (NHEJ) pathway for the benefit of antigen receptor gene diversity. Here, we review the various parameters that constrain repair of RAG-generated DSBs to NHEJ, including the peculiarity of DNA DSB ends generated by the RAG nuclease, the establishment and maintenance of a post-cleavage synaptic complex, and the protection of DNA ends against resection and (micro)homology-directed repair. In this physiological context, we highlight that certain DSBs have limited DNA repair pathway choice options.

scholarly journals Modulation of Genome Editing Outcomes by Cell Cycle Control of Cas9 Expression

2017 ◽  
Author(s):  
Yuping Huang ◽  
Caitlin McCann ◽  
Andrey Samsonov ◽  
Dmitry Malkov ◽  
Gregory D Davis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Fluorescent Protein ◽  
Cell Cycle Control ◽  
Strand Break ◽  
End Joining ◽  
Genetic Changes ◽  
A Cell ◽  
Non Homologous End Joining ◽  
Green Fluorescent ◽  
Chromosomal Sequences

ABSTRACTTargeting specific chromosomal sequences for genome modification or regulation during particular phases of the cell cycle may prove useful in creating more precise, predictable genetic changes. Here, we present a system using a fusion protein comprised of a programmable DNA modification protein, Cas9, linked to a cell cycle regulated protein, geminin, as well as green fluorescent protein (GFP) for visualization. Despite the large size of Cas9 relative to geminin, cells were observed to express Cas9-GFP-geminin at levels which oscillate with the cell cycle. These fusion proteins are also shown to retain double-strand break (DSB) activity at specific chromosomal sequences to produce both indels and targeted integration of donor ssDNA. Most importantly, the ratio of ssDNA donor integration to non-homologous end joining (NHEJ) was observed to increase, suggesting that cell cycle control Cas9 expression may be an effective strategy to bias DNA repair outcomes.

scholarly journals A homology independent sequence replacement strategy in human cells using a CRISPR nuclease

Open Biology ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 200283
Author(s):  
Eric Danner ◽  
Mikhail Lebedin ◽  
Kathrin de la Rosa ◽  
Ralf Kühn
Keyword(s):  
Basic Research ◽  
Strand Break ◽  
Human Cells ◽  
End Joining ◽  
Linear Amplification ◽  
Promising Alternative ◽  
Independent Sequence ◽  
Homology Directed Repair ◽  
Dividing Cells ◽  
Non Homologous End Joining

Precision genomic alterations largely rely on homology directed repair (HDR), but targeting without homology using the non-homologous end-joining (NHEJ) pathway has gained attention as a promising alternative. Previous studies demonstrated precise insertions formed by the ligation of donor DNA into a targeted genomic double-strand break in both dividing and non-dividing cells. Here, we demonstrate the use of NHEJ repair to replace genomic segments with donor sequences; we name this method ‘Replace’ editing ( R ational e nd-joining p rotocol de l ivering a targeted sequen c e e xchange). Using CRISPR/Cas9, we create two genomic breaks and ligate a donor sequence in-between. This exchange of a genomic for a donor sequence uses neither microhomology nor homology arms. We target four loci in cell lines and show successful exchange of exons in 16–54% of human cells. Using linear amplification methods and deep sequencing, we quantify the diversity of outcomes following Replace editing and profile the ligated interfaces. The ability to replace exons or other genomic sequences in cells not efficiently modified by HDR holds promise for both basic research and medicine.

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