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Inactivation of Pol θ and C-NHEJ eliminates off-target integration of exogenous DNA

Published on Dec 1, 2017in Nature Communications11.88
· DOI :10.1038/s41467-017-00124-3
Alexander Zelensky4
Estimated H-index: 4
(EUR: Erasmus University Rotterdam),
Joost Schimmel1
Estimated H-index: 1
(LEI: Leiden University)
+ 2 AuthorsMarcel Tijsterman30
Estimated H-index: 30
(LEI: Leiden University)
Cite
Abstract
Off-target or random integration of exogenous DNA hampers precise genomic engineering and presents a safety risk in clinical gene therapy strategies. Genetic definition of random integration has been lacking for decades. Here, we show that the A-family DNA polymerase θ (Pol θ) promotes random integration, while canonical non-homologous DNA end joining plays a secondary role; cells double deficient for polymerase θ and canonical non-homologous DNA end joining are devoid of any integration events, demonstrating that these two mechanisms define random integration. In contrast, homologous recombination is not reduced in these cells and gene targeting is improved to 100% efficiency. Such complete reversal of integration outcome, from predominately random integration to exclusively gene targeting, provides a rational way forward to improve the efficacy and safety of DNA delivery and gene correction approaches. Random off-target integration events can impair precise gene targeting and poses a safety risk for gene therapy. Here the authors show that repression of polymerase θ and classical non-homologous recombination eliminates random integration.
  • References (45)
  • Citations (20)
Cite
References45
Newest
Published on Dec 1, 2017in Nature Communications11.88
Shinta Saito4
Estimated H-index: 4
,
Ryo Maeda4
Estimated H-index: 4
,
Noritaka Adachi29
Estimated H-index: 29
Homologous recombination mediated gene targeting is highly inefficient in human cells due to random integration events, Here the authors show that dual repression of polymerase θ and DNA ligase IV eliminate random integration events.
Published on Dec 1, 2016in Journal of Controlled Release7.90
Jia Liu12
Estimated H-index: 12
(ShanghaiTech University),
Sai-lan Shui4
Estimated H-index: 4
(ShanghaiTech University)
The advent of site-specific nucleases, particularly CRISPR/Cas9, provides researchers with the unprecedented ability to manipulate genomic sequences. These nucleases are used to create model cell lines, engineer metabolic pathways, produce transgenic animals and plants, perform genome-wide functional screen and, most importantly, treat human diseases that are difficult to tackle by traditional medications. Considerable efforts have been devoted to improving the efficiency and specificity of nucl...
Published on Nov 1, 2016in Nature plants13.30
Maartje van Kregten2
Estimated H-index: 2
,
Sylvia de Pater12
Estimated H-index: 12
+ 3 AuthorsMarcel Tijsterman30
Estimated H-index: 30
The mechanism for T-DNA integration, a critical step of Agrobacterium-mediated transgenesis, remains elusive. Now, a study shows that polymerase θ controls T-DNA integration and generates the error-prone sequences at the sites of integration.
Published on Sep 1, 2016in Theoretical and Applied Genetics3.93
Ayushi Kamthan5
Estimated H-index: 5
,
Abira Chaudhuri3
Estimated H-index: 3
+ 1 AuthorsAsis Datta27
Estimated H-index: 27
Key message New advances in crop genetic engineering can significantly pace up the development of genetically improved varieties with enhanced yield, nutrition and tolerance to biotic and abiotic stresses.
Published on Jul 8, 2016in Nucleic Acids Research11.15
Sara Ahrabi3
Estimated H-index: 3
(University of Oxford),
Sovan Sarkar9
Estimated H-index: 9
(University of Oxford)
+ 4 AuthorsTimothy C. Humphrey11
Estimated H-index: 11
(University of Oxford)
DNA double-strand breaks (DSBs) are toxic lesions, which if improperly repaired can result in cell death or genomic instability. DSB repair is usually facilitated by the classical non-homologous end joining (C-NHEJ), or homologous recombination (HR) pathways. However, a mutagenic alternative NHEJ pathway, microhomology-mediated end joining (MMEJ), can also be deployed. While MMEJ is suppressed by C-NHEJ, the relationship between HR and MMEJ is less clear. Here, we describe a role for HR genes in...
Published on Feb 29, 2016in Nucleic Acids Research11.15
Gemael-Cedrick Taty-Taty2
Estimated H-index: 2
(University of Toulouse),
Catherine Chailleux7
Estimated H-index: 7
(University of Toulouse)
+ 6 AuthorsYvan Canitrot14
Estimated H-index: 14
(University of Toulouse)
Repair of DNA double-strand breaks occurs in a chromatin context that needs to be modified and remodeled to allow suitable access to the different DNA repair machineries. Of particular importance for the maintenance of genetic stability is the tight control of error-prone pathways, such as the alternative End Joining pathway. Here, we show that the chromatin remodeler p400 ATPase is a brake to the use of alternative End Joining. Using specific intracellular reporter susbstrates we observed that ...
Shahnaz Masani5
Estimated H-index: 5
(MSU: Michigan State University),
Li Han10
Estimated H-index: 10
(MSU: Michigan State University)
+ 1 AuthorsKefei Yu20
Estimated H-index: 20
(MSU: Michigan State University)
Abstract Nonhomologous end-joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammals and resolves the DSBs generated during both V(D)J recombination in developing lymphocytes and class switch recombination (CSR) in antigen-stimulated B cells. In contrast to the absolute requirement for NHEJ to resolve DSBs associated with V(D)J recombination, DSBs associated with CSR can be resolved in NHEJ-deficient cells (albeit at a reduced level) by a poorly defined alternative end-...
Guangqing Lu4
Estimated H-index: 4
(Peking Union Medical College),
Jinzhi Duan4
Estimated H-index: 4
(Peking Union Medical College)
+ 4 AuthorsYu Zhang43
Estimated H-index: 43
(Peking Union Medical College)
Abstract In eukaryotes, DNA double-strand breaks (DSBs), one of the most harmful types of DNA damage, are repaired by homologous repair (HR) and nonhomologous end-joining (NHEJ). Surprisingly, in cells deficient for core classic NHEJ factors such as DNA ligase IV (Lig4), substantial end-joining activities have been observed in various situations, suggesting the existence of alternative end-joining (A-EJ) activities. Several putative A-EJ factors have been proposed, although results are mostly co...
Published on Jan 1, 2016in Biological & Pharmaceutical Bulletin1.54
Shinta Saito4
Estimated H-index: 4
(YCU: Yokohama City University),
Noritaka Adachi29
Estimated H-index: 29
(YCU: Yokohama City University)
Published on Feb 1, 2015in Nature43.07
Raphael Ceccaldi12
Estimated H-index: 12
,
Jessica C. Liu2
Estimated H-index: 2
+ 9 AuthorsSimon J. Boulton47
Estimated H-index: 47
In studies in mammalian cells, polymerase theta (Polθ, also known as POLQ) is identified as the polymerase responsible for non-homologous end joining DNA repair; this DNA repair pathway acts in many tumours when homologous recombination is inactivated and the identification of the polymerase responsible may aid the development of new therapeutic approaches.
Cited By20
Newest
Published on Mar 8, 2019in Nature Communications11.88
Grégoire Cullot (French Institute of Health and Medical Research), Julian Boutin (French Institute of Health and Medical Research)+ 17 AuthorsVéronique Guyonnet-Duperat12
Estimated H-index: 12
(French Institute of Health and Medical Research)
CRISPR-Cas9 is a promising technology for genome editing. Here we use Cas9 nuclease-induced double-strand break DNA (DSB) at the UROS locus to model and correct congenital erythropoietic porphyria. We demonstrate that homology-directed repair is rare compared with NHEJ pathway leading to on-target indels and causing unwanted dysfunctional protein. Moreover, we describe unexpected chromosomal truncations resulting from only one Cas9 nuclease-induced DSB in cell lines and primary cells by a p53-de...
Published on Mar 11, 2019in Earth, Planets and Space2.74
Masaya Kimura1
Estimated H-index: 1
(UTokyo: University of Tokyo),
Nobuki Kame11
Estimated H-index: 11
(UTokyo: University of Tokyo)
+ 5 AuthorsTakashi Kunugi12
Estimated H-index: 12
Earthquake ruptures cause mass redistribution, which is expected to induce transient gravity perturbations simultaneously at all distances from the source before the arrival of P-waves. A recent research paper reported the detection of such prompt gravity signals from the 2011 Tohoku-Oki earthquake by comparing observed acceleration waveforms and model simulations. The 11 observed waveforms presented in that paper recorded in East Asia shared a similar trend above the background seismic noise an...
Published on 2019in Nature Communications11.88
Tarun S. Nambiar (Columbia University), Pierre Billon (Columbia University)+ 7 AuthorsAndrew Palacios (Columbia University)
Precise editing of genomic DNA can be achieved upon repair of CRISPR-induced DNA double-stranded breaks (DSBs) by homology-directed repair (HDR). However, the efficiency of this process is limited by DSB repair pathways competing with HDR, such as non-homologous end joining (NHEJ). Here we individually express in human cells 204 open reading frames involved in the DNA damage response (DDR) and determine their impact on CRISPR-mediated HDR. From these studies, we identify RAD18 as a stimulator of...
Published on Dec 1, 2019in Nature Communications11.88
Wanjuan Feng2
Estimated H-index: 2
(UNC: University of North Carolina at Chapel Hill),
Dennis A. Simpson18
Estimated H-index: 18
(UNC: University of North Carolina at Chapel Hill)
+ 9 AuthorsJoel S. Parker73
Estimated H-index: 73
(UNC: University of North Carolina at Chapel Hill)
Polymerase theta (Pol θ, gene name Polq) is a widely conserved DNA polymerase that mediates a microhomology-mediated, error-prone, double strand break (DSB) repair pathway, referred to as Theta Mediated End Joining (TMEJ). Cells with homologous recombination deficiency are reliant on TMEJ for DSB repair. It is unknown whether deficiencies in other components of the DNA damage response (DDR) also result in Pol θ addiction. Here we use a CRISPR genetic screen to uncover 140 Polq synthetic lethal (...
Published on 2019in DNA Repair3.71
Sandra Bosshard2
Estimated H-index: 2
(UNIL: University of Lausanne),
Pierre-Olivier Duroy (UNIL: University of Lausanne), Nicolas Mermod30
Estimated H-index: 30
(UNIL: University of Lausanne)
Abstract CRISPR technologies greatly foster genome editing in mammalian cells through site-directed DNA double strand breaks (DSBs). However, precise editing outcomes, as mediated by homologous recombination (HR) repair, are typically infrequent and outnumbered by undesired genome alterations. By using knockdown and overexpression studies in Chinese hamster ovary (CHO) cells as well as characterizing repaired DNA junctions, we found that efficient HR-mediated genome editing depends on alternativ...
Published on Jul 1, 2019in Trends in Genetics10.63
Joost Schimmel8
Estimated H-index: 8
(LUMC: Leiden University Medical Center),
Robin van Schendel9
Estimated H-index: 9
(LUMC: Leiden University Medical Center)
+ 1 AuthorsMarcel Tijsterman30
Estimated H-index: 30
(LUMC: Leiden University Medical Center)
A recognized source of disease-causing genome alterations is erroneous repair of broken chromosomes, which can be executed by two distinct mechanisms: non-homologous end joining (NHEJ) and the recently discovered polymerase theta-mediated end joining (TMEJ) pathway. While TMEJ has previously been considered to act as an alternative mechanism backing up NHEJ, recent work points to a role for TMEJ in the repair of replication-associated DNA breaks that are excluded from repair through homologous r...
Seuk-Min Ryu (KIST: Korea Institute of Science and Technology), Junseok W. Hur6
Estimated H-index: 6
(KU: Korea University),
Kyoungmi Kim (KU: Korea University)
Published on 2019in PLOS Genetics5.22
Andrew A. Kelso (Beckman Research Institute), Felicia Wednesday Lopezcolorado (Beckman Research Institute)+ 1 AuthorsJeremy M. Stark9
Estimated H-index: 9
(Beckman Research Institute)
Disrupting either the DNA annealing factor RAD52 or the A-family DNA polymerase POLQ can cause synthetic lethality with defects in BRCA1 and BRCA2, which are tumor suppressors important for homology-directed repair of DNA double-strand breaks (DSBs), and protection of stalled replication forks. A likely mechanism of this synthetic lethality is that RAD52 and/or POLQ are important for backup pathways for DSB repair and/or replication stress responses. The features of DSB repair events that requir...
Published on Jun 12, 2019in Nature43.07
Sanne E. Klompe (Columbia University), Phuc L. H. Vo (Columbia University)+ 1 AuthorsSamuel H. Sternberg20
Estimated H-index: 20
(Columbia University)
Conventional CRISPR–Cas systems maintain genomic integrity by leveraging guide RNAs for the nuclease-dependent degradation of mobile genetic elements, including plasmids and viruses. Here we describe a remarkable inversion of this paradigm, in which bacterial Tn7-like transposons have co-opted nuclease-deficient CRISPR–Cas systems to catalyze RNA-guided integration of mobile genetic elements into the genome. Programmable transposition of Vibrio cholerae Tn6677 in E. coli requires CRISPR- and tra...
Published on Jun 1, 2019in Cell Reports7.82
Inger Brandsma8
Estimated H-index: 8
(Erasmus University Medical Center),
Koichi Sato (UU: Utrecht University)+ 17 AuthorsKarel Bezstarosti26
Estimated H-index: 26
(Erasmus University Medical Center)
Summary The tumor suppressor BRCA2 is essential for homologous recombination (HR), replication fork stability, and DNA interstrand crosslink repair in vertebrates. We identify HSF2BP, a protein previously described as testis specific and not characterized functionally, as an interactor of BRCA2 in mouse embryonic stem cells, where the 2 proteins form a constitutive complex. HSF2BP is transcribed in all cultured human cancer cell lines tested and elevated in some tumor samples. Inactivation of th...
View next paperDual loss of human POLQ and LIG4 abolishes random integration