scholarly journals Methods Favoring Homology-Directed Repair Choice in Response to CRISPR/Cas9 Induced-Double Strand Breaks

2020 ◽  
Vol 21 (18) ◽  
pp. 6461
Author(s):  
Han Yang ◽  
Shuling Ren ◽  
Siyuan Yu ◽  
Haifeng Pan ◽  
Tingdong Li ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Initial Step ◽  
Double Strand Breaks ◽  
Repair Pathway ◽  
End Joining ◽  
Strand Breaks ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Repair Pathways

Precise gene editing is—or will soon be—in clinical use for several diseases, and more applications are under development. The programmable nuclease Cas9, directed by a single-guide RNA (sgRNA), can introduce double-strand breaks (DSBs) in target sites of genomic DNA, which constitutes the initial step of gene editing using this novel technology. In mammals, two pathways dominate the repair of the DSBs—nonhomologous end joining (NHEJ) and homology-directed repair (HDR)—and the outcome of gene editing mainly depends on the choice between these two repair pathways. Although HDR is attractive for its high fidelity, the choice of repair pathway is biased in a biological context. Mammalian cells preferentially employ NHEJ over HDR through several mechanisms: NHEJ is active throughout the cell cycle, whereas HDR is restricted to S/G2 phases; NHEJ is faster than HDR; and NHEJ suppresses the HDR process. This suggests that definitive control of outcome of the programmed DNA lesioning could be achieved through manipulating the choice of cellular repair pathway. In this review, we summarize the DSB repair pathways, the mechanisms involved in choice selection based on DNA resection, and make progress in the research investigating strategies that favor Cas9-mediated HDR based on the manipulation of repair pathway choice to increase the frequency of HDR in mammalian cells. The remaining problems in improving HDR efficiency are also discussed. This review should facilitate the development of CRISPR/Cas9 technology to achieve more precise gene editing.

scholarly journals The Determinant of DNA Repair Pathway Choices in Ionising Radiation-Induced DNA Double-Strand Breaks

2020 ◽  
Vol 2020 ◽  
pp. 1-12 ◽  
Author(s):  
Lei Zhao ◽  
Chengyu Bao ◽  
Yuxuan Shang ◽  
Xinye He ◽  
Chiyuan Ma ◽  
...  
Keyword(s):  
Dna Repair ◽  
Double Strand Breaks ◽  
Ionising Radiation ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Dna Dsbs ◽  
Repair Pathways

Ionising radiation- (IR-) induced DNA double-strand breaks (DSBs) are considered to be the deleterious DNA lesions that pose a serious threat to genomic stability. The major DNA repair pathways, including classical nonhomologous end joining, homologous recombination, single-strand annealing, and alternative end joining, play critical roles in countering and eliciting IR-induced DSBs to ensure genome integrity. If the IR-induced DNA DSBs are not repaired correctly, the residual or incorrectly repaired DSBs can result in genomic instability that is associated with certain human diseases. Although many efforts have been made in investigating the major mechanisms of IR-induced DNA DSB repair, it is still unclear what determines the choices of IR-induced DNA DSB repair pathways. In this review, we discuss how the mechanisms of IR-induced DSB repair pathway choices can operate in irradiated cells. We first briefly describe the main mechanisms of the major DNA DSB repair pathways and the related key repair proteins. Based on our understanding of the characteristics of IR-induced DNA DSBs and the regulatory mechanisms of DSB repair pathways in irradiated cells and recent advances in this field, We then highlight the main factors and associated challenges to determine the IR-induced DSB repair pathway choices. We conclude that the type and distribution of IR-induced DSBs, chromatin state, DNA-end structure, and DNA-end resection are the main determinants of the choice of the IR-induced DNA DSB repair pathway.

scholarly journals Homology Directed Repair by Cas9:Donor Co-localization in Mammalian Cells

2018 ◽  
Author(s):  
Philip J.R. Roche ◽  
Heidi Gytz ◽  
Faiz Hussain ◽  
Christopher J.F. Cameron ◽  
Denis Paquette ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Low Frequency ◽  
Cost Effective ◽  
Hek293 Cells ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

AbstractHomology directed repair (HDR) induced by site specific DNA double strand breaks (DSB) with CRISPR/Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered Cas9 protein fused to a high-affinity monoavidin domain to deliver biotinylated donor DNA to a DSB site. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9 RNP-donor DNA complex into the cell. Combining these strategies improved HDR percentages of up to 90% in three tested loci (CXCR4, EMX1, and TLR) in standard HEK293 cells. Our approach offers a cost effective, simple and broadly applicable gene editing method, thereby expanding the CRISPR/Cas9 genome editing toolbox.SummaryPrecision gene editing occurs at a low percentage in mammalian cells using Cas9. Colocalization of donor with Cas9MAV and PEI delivery raises HDR occurrence.

scholarly journals Engineering a far-red light–activated split-Cas9 system for remote-controlled genome editing of internal organs and tumors

2020 ◽  
Vol 6 (28) ◽  
pp. eabb1777 ◽  
Author(s):  
Yuanhuan Yu ◽  
Xin Wu ◽  
Ningzi Guan ◽  
Jiawei Shao ◽  
Huiying Li ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Light Emitting Diode ◽  
Red Light ◽  
Tumor Model ◽  
End Joining ◽  
Internal Organs ◽  
Fast System ◽  
Homology Directed Repair ◽  
Multiple Loci

It is widely understood that CRISPR-Cas9 technology is revolutionary, with well-recognized issues including the potential for off-target edits and the attendant need for spatiotemporal control of editing. Here, we describe a far-red light (FRL)–activated split-Cas9 (FAST) system that can robustly induce gene editing in both mammalian cells and mice. Through light-emitting diode–based FRL illumination, the FAST system can efficiently edit genes, including nonhomologous end joining and homology-directed repair, for multiple loci in human cells. Further, we show that FAST readily achieves FRL-induced editing of internal organs in tdTomato reporter mice. Finally, FAST was demonstrated to achieve FRL-triggered editing of the PLK1 oncogene in a mouse xenograft tumor model. Beyond extending the spectrum of light energies in optogenetic toolbox for CRISPR-Cas9 technologies, this study demonstrates how FAST system can be deployed for programmable deep tissue gene editing in both biological and biomedical contexts toward high precision and spatial specificity.

scholarly journals DNA polymerases δ and λ cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae

2015 ◽  
Vol 112 (50) ◽  
pp. E6907-E6916 ◽  
Author(s):  
Damon Meyer ◽  
Becky Xu Hua Fu ◽  
Wolf-Dietrich Heyer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Polymerase ◽  
Genome Stability ◽  
Double Strand Breaks ◽  
Dna Polymerase Delta ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Repair Efficiency ◽  
Repair Pathways

Maintenance of genome stability is carried out by a suite of DNA repair pathways that ensure the repair of damaged DNA and faithful replication of the genome. Of particular importance are the repair pathways, which respond to DNA double-strand breaks (DSBs), and how the efficiency of repair is influenced by sequence homology. In this study, we developed a genetic assay in diploid Saccharomyces cerevisiae cells to analyze DSBs requiring microhomologies for repair, known as microhomology-mediated end-joining (MMEJ). MMEJ repair efficiency increased concomitant with microhomology length and decreased upon introduction of mismatches. The central proteins in homologous recombination (HR), Rad52 and Rad51, suppressed MMEJ in this system, suggesting a competition between HR and MMEJ for the repair of a DSB. Importantly, we found that DNA polymerase delta (Pol δ) is critical for MMEJ, independent of microhomology length and base-pairing continuity. MMEJ recombinants showed evidence that Pol δ proofreading function is active during MMEJ-mediated DSB repair. Furthermore, mutations in Pol δ and DNA polymerase 4 (Pol λ), the DNA polymerase previously implicated in MMEJ, cause a synergistic decrease in MMEJ repair. Pol λ showed faster kinetics associating with MMEJ substrates following DSB induction than Pol δ. The association of Pol δ depended on RAD1, which encodes the flap endonuclease needed to cleave MMEJ intermediates before DNA synthesis. Moreover, Pol δ recruitment was diminished in cells lacking Pol λ. These data suggest cooperative involvement of both polymerases in MMEJ.

Regulation of the cellular DNA double-strand break response

2007 ◽  
Vol 85 (6) ◽  
pp. 663-674 ◽  
Author(s):  
Kendra L. Cann ◽  
Geoffrey G. Hicks
Keyword(s):  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Cell Cycle Checkpoints ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Cellular Dna

DNA double-strand breaks occur frequently in cycling cells, and are also induced by exogenous sources, including ionizing radiation. Cells have developed integrated double-strand break response pathways to cope with these lesions, including pathways that initiate DNA repair (either via homologous recombination or nonhomologous end joining), the cell-cycle checkpoints (G1–S, intra-S phase, and G2–M) that provide time for repair, and apoptosis. However, before any of these pathways can be activated, the damage must first be recognized. In this review, we will discuss how the response of mammalian cells to DNA double-strand breaks is regulated, beginning with the activation of ATM, the pinnacle kinase of the double-strand break signalling cascade.

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.

Double-Strand Breaks Repair by Non-Homologous DNA End Joining in Mammalian Cells

2006 ◽  
Vol 7 (5) ◽  
pp. 311-322 ◽  
Author(s):  
Mariusz Malinowski ◽  
Elzbieta Pastwa
Keyword(s):  
Mammalian Cells ◽  
Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks

Repairing DNA double-strand breaks by the prokaryotic non-homologous end-joining pathway

2009 ◽  
Vol 37 (3) ◽  
pp. 539-545 ◽  
Author(s):  
Nigel C. Brissett ◽  
Aidan J. Doherty
Keyword(s):  
Molecular Mechanisms ◽  
Model System ◽  
Double Strand Breaks ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Non Homologous End Joining ◽  
Eukaryotic Organisms

The NHEJ (non-homologous end-joining) pathway is one of the major mechanisms for repairing DSBs (double-strand breaks) that occur in genomic DNA. In common with eukaryotic organisms, many prokaryotes possess a conserved NHEJ apparatus that is essential for the repair of DSBs arising in the stationary phase of the cell cycle. Although the bacterial NHEJ complex is much more minimal than its eukaryotic counterpart, both pathways share a number of common mechanistic features. The relative simplicity of the prokaryotic NHEJ complex makes it a tractable model system for investigating the cellular and molecular mechanisms of DSB repair. The present review describes recent advances in our understanding of prokaryotic end-joining, focusing primarily on biochemical, structural and cellular aspects of the mycobacterial NHEJ repair pathway.

Sign in / Sign up

Export Citation Format

Share Document