The Experts below are selected from a list of 4776 Experts worldwide ranked by ideXlab platform
Takashi Yamamoto - One of the best experts on this subject based on the ideXlab platform.
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A Simple Knock-In System for Xenopus via Microhomology Mediated End Joining Repair.
Methods in molecular biology (Clifton N.J.), 2018Co-Authors: Ken Ich T Suzuki, Yuto Sakane, Miyuki Suzuki, Takashi YamamotoAbstract:Following completion of the genome sequences of Xenopus tropicalis and X. laevis, gene targeting techniques have become increasingly important for the further development of Xenopus research in the life sciences. Gene knockout using programmable nucleases, such as TALEN and CRISPR/Cas9, has reached a level whereby we can readily and routinely perform loss-of-function analysis of genes of interest in these species. However, there is still room for improvement in gene knock-in techniques owing to some technical problems. To overcome these problems, several knock-in techniques have been developed. Among them, we introduce in this chapter a simple knock-in system mediated by microhomology mediated End Joining repair. This protocol allows us to produce knock-in animals for in vivo tagging, promoter/enhancer traps, and transgenesis in both of these Xenopus species.
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Biased genome editing using the local accumulation of DSB repair molecules system
Nature communications, 2018Co-Authors: Shota Nakade, Tomomi Aida, Tetsushi Sakuma, Keiji Mochida, Kohichi Tanaka, Naoaki Sakamoto, Atsushi Kunii, Kazuki Nakamae, Takashi YamamotoAbstract:Selective genome editing such as gene knock-in has recently been achieved by administration of chemical enhancer or inhibitor of particular DNA double-strand break (DSB) repair pathways, as well as overexpression of pathway-specific genes. In this study, we attempt to enhance the efficiency further to secure robust gene knock-ins, by using the local accumulation of DSB repair molecules (LoAD) system. We identify CtIP as a strong enhancer of Microhomology-Mediated End-Joining (MMEJ) repair by genetic screening, and show the knock-in-enhancing effect of CtIP LoADing. Next-generation sequencing reveals that CtIP LoADing highly increases the frequency of MMEJ-mediated integration. Selection-free, simultaneous triple gene knock-ins are also achieved with the CtIP-LoADing strategy. Moreover, by replacing the LoADing molecules and targeting strategies, this system can be applied for other specific genome engineering purposes, such as introducing longer deletions for gene disruption, indepEndently introducing multiple mutations without chromosomal deletion, and efficiently incorporating a single-stranded oligodeoxynucleotide donor. Genome editing using CRISPR can be enhanced by manipulating DNA double-strand break repair pathways. Here the authors demonstrate LoAD, local accumulation of repair molecules, which shifts repair to Microhomology-Mediated End-Joining.
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Microhomology-assisted scarless genome editing in human iPSCs
Nature communications, 2018Co-Authors: Shin Il Kim, Tetsushi Sakuma, Tomoko Matsumoto, Harunobu Kagawa, Michiko Nakamura, Ryoko Hirohata, Ayano Ueno, Maki Ohishi, Tomoyoshi Soga, Takashi YamamotoAbstract:Gene-edited induced pluripotent stem cells (iPSCs) provide relevant isogenic human disease models in patient-specific or healthy genetic backgrounds. Towards this End, gene targeting using antibiotic selection along with engineered point mutations remains a reliable method to enrich edited cells. Nevertheless, integrated selection markers obstruct scarless transgene-free gene editing. Here, we present a method for scarless selection marker excision using engineered Microhomology-Mediated End Joining (MMEJ). By overlapping the homology arms of standard donor vectors, short tandem microhomologies are generated flanking the selection marker. Unique CRISPR-Cas9 protospacer sequences nested between the selection marker and engineered microhomologies are cleaved after gene targeting, engaging MMEJ and scarless excision. Moreover, when point mutations are positioned unilaterally within engineered microhomologies, both mutant and normal isogenic clones are derived simultaneously. The utility and fidelity of our method is demonstrated in human iPSCs by editing the X-linked HPRT1 locus and biallelic modification of the autosomal APRT locus, eliciting disease-relevant metabolic phenotypes.
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Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ
BMC Genomics, 2016Co-Authors: Tomomi Aida, Shota Nakade, Yayoi Izu, Ayu Oishi, Hidenori Aizawa, Tetsushi Sakuma, Harumi Ishikubo, Keiji Mochida, Takako Usami, Takashi YamamotoAbstract:Although CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by Microhomology-Mediated End-Joining. Here, we identified exonuclease 1 (Exo1) as an enhancer for PITCh in human cells. By combining the Exo1 and PITCh-directed donor vectors, we achieved convenient one-step knock-in of gene cassettes and floxed allele both in human cells and mouse zygotes. Our results provide a technical platform for high-throughput knock-in.
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Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ
BMC Genomics, 2016Co-Authors: Tomomi Aida, Shota Nakade, Ayu Oishi, Hidenori Aizawa, Tetsushi Sakuma, Harumi Ishikubo, Keiji Mochida, Takako Usami, Takashi Yamamoto, Kohichi TanakaAbstract:Background Although CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by Microhomology-Mediated End-Joining. Results Here, we identified exonuclease 1 ( Exo1 ) as an enhancer for PITCh in human cells. By combining the Exo1 and PITCh-directed donor vectors, we achieved convenient one-step knock-in of gene cassettes and floxed allele both in human cells and mouse zygotes. Conclusions Our results provide a technical platform for high-throughput knock-in.
Sang Eun Lee - One of the best experts on this subject based on the ideXlab platform.
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Microhomology Selection for Microhomology Mediated End Joining in Saccharomyces cerevisiae.
Genes, 2019Co-Authors: Kihoon Lee, Ja Hwan Seol, Sang Eun Lee, Kihoon Yoon, Jun Che, Eun Yong ShimAbstract:Microhomology-Mediated End Joining (MMEJ) anneals short, imperfect microhomologies flanking DNA breaks, producing repair products with deletions in a Ku- and RAD52-indepEndent fashion. Puzzlingly, MMEJ preferentially selects certain microhomologies over others, even when multiple microhomologies are available. To define rules and parameters for microhomology selection, we altered the length, the position, and the level of mismatches to the microhomologies flanking homothallic switching (HO) Endonuclease-induced breaks and assessed their effect on MMEJ frequency and the types of repair product formation. We found that microhomology of eight to 20 base pairs carrying no more than 20% mismatches efficiently induced MMEJ. Deletion of MSH6 did not impact MMEJ frequency. MMEJ preferentially chose a microhomology pair that was more proximal from the break. Interestingly, MMEJ events preferentially retained the centromere proximal side of the HO break, while the sequences proximal to the telomere were frequently deleted. The asymmetry in the deletional profile among MMEJ products was reduced when HO was induced on the circular chromosome. The results provide insight into how cells search and select microhomologies for MMEJ in budding yeast.
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Microhomology-Mediated End Joining: Good, bad and ugly.
Mutation research, 2017Co-Authors: Ja Hwan Seol, Eun Yong Shim, Sang Eun LeeAbstract:DNA double-strand breaks (DSBs) are induced by a variety of genotoxic agents, including ionizing radiation and chemotherapy drugs for treating cancers. The elimination of DSBs proceeds via distinctive error-free and error-prone pathways. Repair by homologous recombination (HR) is largely error-free and mediated by RAD51/BRCA2 gene products. Classical non-homologous End Joining (C-NHEJ) requires the Ku heterodimer and can efficiently rejoin breaks, with occasional loss or gain of DNA information. Recently, evidence has unveiled another DNA End-Joining mechanism that is indepEndent of recombination factors and Ku proteins, termed alternative non-homologous End Joining (A-NHEJ). While A-NHEJ-mediated repair does not require homology, in a subtype of A-NHEJ, DSB breaks are sealed by microhomology (MH)-mediated base-pairing of DNA single strands, followed by nucleolytic trimming of DNA flaps, DNA gap filling, and DNA ligation, yielding products that are always associated with DNA deletion. This highly error-prone DSB repair pathway is termed Microhomology-Mediated End Joining (MMEJ). Dissecting the mechanisms of MMEJ is of great interest because of its potential to destabilize the genome through gene deletions and chromosomal rearrangements in cells deficient in canonical repair pathways, including HR and C-NHEJ. In addition, evidence now suggests that MMEJ plays a physiological role in normal cells.
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Microhomology-Mediated End Joining induces hypermutagenesis at breakpoint junctions.
PLoS genetics, 2017Co-Authors: Supriya Sinha, Eun Yong Shim, Diana D. Villarreal, Jae Hoon Shim, Suhyeon Yoon, Kyungjae Myung, Sang Eun LeeAbstract:Microhomology (MH) flanking a DNA double-strand break (DSB) drives chromosomal rearrangements but its role in mutagenesis has not yet been analyzed. Here we determined the mutation frequency of a URA3 reporter gene placed at multiple locations distal to a DSB, which is flanked by different sizes (15-, 18-, or 203-bp) of direct repeat sequences for efficient repair in budding yeast. Induction of a DSB accumulates mutations in the reporter gene situated up to 14-kb distal to the 15-bp MH, but more modestly to those carrying 18- and 203-bp or no homology. Increased mutagenesis in MH-mediated End Joining (MMEJ) appears coupled to its slower repair kinetics and the extensive resection occurring at flanking DNA. Chromosomal translocations via MMEJ also elevate mutagenesis of the flanking DNA sequences 7.1 kb distal to the breakpoint junction as compared to those without MH. The results suggest that MMEJ could destabilize genomes by triggering structural alterations and increasing mutation burden.
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Risky business: Microhomology-Mediated End Joining.
Mutation research, 2016Co-Authors: Supriya Sinha, Eun Yong Shim, Diana D. Villarreal, Sang Eun LeeAbstract:Prevalence of microhomology (MH) at the breakpoint junctions in somatic and germ-line chromosomal rearrangements and in the programmed immune receptor rearrangements from cells deficient in classical End Joining reveals an enigmatic process called MH-mediated End Joining (MMEJ). MMEJ repairs DNA double strand breaks (DSBs) by annealing flanking MH and deleting genetic information at the repair junctions from yeast to humans. Being genetically distinct from canonical DNA DSB pathways, MMEJ is involved with the fusions of eroded/uncapped telomeres as well as with the assembly of chromosome fragments in chromothripsis. In this review article, we will discuss an up-to-date model representing the MMEJ process and the mechanism by which cells regulate MMEJ to limit repair-associated mutagenesis. We will also describe the possible therapeutic gains resulting from the inhibition of MMEJ in recombination deficient cancers. Lastly, we will embark on two contentious issues associated with MMEJ such as the significance of MH at the repair junction to be the hallmark of MMEJ and the relationship of MMEJ to other mechanistically related DSB repair pathways.
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MMEJ repair of double-strand breaks (director’s cut): deleted sequences and alternative Endings
Trends in genetics : TIG, 2008Co-Authors: Mitch Mcvey, Sang Eun LeeAbstract:DNA double-strand breaks are normal consequences of cell division and differentiation and must be repaired faithfully to maintain genome stability. Two mechanistically distinct pathways are known to efficiently repair double-strand breaks: homologous recombination and Ku-depEndent non-homologous End Joining. Recently, a third, less characterized repair mechanism, named Microhomology-Mediated End Joining (MMEJ), has received increasing attention. MMEJ repairs DNA breaks via the use of substantial microhomology and always results in deletions. Furthermore, it probably contributes to oncogenic chromosome rearrangements and genetic variation in humans. Here, we summarize the genetic attributes of MMEJ from several model systems and discuss the relationship between MMEJ and 'alternative End Joining'. We propose a mechanistic model for MMEJ and highlight important questions for future research.
Mitch Mcvey - One of the best experts on this subject based on the ideXlab platform.
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Drosophila DNA polymerase theta utilizes both helicase-like and polymerase domains during Microhomology-Mediated End Joining and interstrand crosslink repair
PLoS genetics, 2017Co-Authors: Kelly Beagan, Robin L. Armstrong, Alice Witsell, Upasana Roy, Nikolai Renedo, Amy Baker, Orlando D. Schärer, Mitch McveyAbstract:Double strand breaks (DSBs) and interstrand crosslinks (ICLs) are toxic DNA lesions that can be repaired through multiple pathways, some of which involve shared proteins. One of these proteins, DNA Polymerase θ (Pol θ), coordinates a mutagenic DSB repair pathway named Microhomology-Mediated End Joining (MMEJ) and is also a critical component for bypass or repair of ICLs in several organisms. Pol θ contains both polymerase and helicase-like domains that are tethered by an unstructured central region. While the role of the polymerase domain in promoting MMEJ has been studied extensively both in vitro and in vivo, a function for the helicase-like domain, which possesses DNA-depEndent ATPase activity, remains unclear. Here, we utilize genetic and biochemical analyses to examine the roles of the helicase-like and polymerase domains of Drosophila Pol θ. We demonstrate an absolute requirement for both polymerase and ATPase activities during ICL repair in vivo. However, similar to mammalian systems, polymerase activity, but not ATPase activity, is required for ionizing radiation-induced DSB repair. Using a site-specific break repair assay, we show that overall End-Joining efficiency is not affected in ATPase-dead mutants, but there is a significant decrease in templated insertion events. In vitro, Pol θ can efficiently bypass a model unhooked nitrogen mustard crosslink and promote DNA synthesis following microhomology annealing, although ATPase activity is not required for these functions. Together, our data illustrate the functional importance of the helicase-like domain of Pol θ and suggest that its tethering to the polymerase domain is important for its multiple functions in DNA repair and damage tolerance.
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RPA puts the brakes on MMEJ
Nature structural & molecular biology, 2014Co-Authors: Mitch McveyAbstract:Microhomology-Mediated End Joining (MMEJ) is a mechanism of DNA double-strand-break repair that creates deletions and promotes other types of genome instability. New in vivo and in vitro analyses demonstrate that the heterotrimeric replication protein A (RPA) complex prevents spontaneous annealing of microhomologies, thereby preventing genome-destabilizing MMEJ.
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Synthesis-depEndent Microhomology-Mediated End Joining accounts for multiple types of repair junctions
Nucleic acids research, 2010Co-Authors: Mitch McveyAbstract:Ku or DNA ligase 4-indepEndent alternative End Joining (alt-EJ) repair of DNA double-strand breaks (DSBs) frequently correlates with increased junctional microhomology. However, alt-EJ also produces junctions without microhomology (apparent blunt joins), and the exact role of microhomology in both alt-EJ and classical non-homologous End Joining (NHEJ) remains unclear. To better understand the degree to which alt-EJ depEnds on annealing at pre-existing microhomologies, we examined inaccurate repair of an I-SceI DSB lacking nearby microhomologies of greater than four nucleotides in Drosophila. Lig4 deficiency affected neither frequency nor length of junctional microhomology, but significantly increased insertion frequency. Many insertions appeared to be templated. Based on sequence analysis of repair junctions, we propose a model of synthesis-depEndent Microhomology-Mediated End Joining (SD-MMEJ), in which de novo synthesis by an accurate non-processive DNA polymerase creates microhomology. Repair junctions with apparent blunt joins, junctional microhomologies and short indels (deletion with insertion) are often considered to reflect different repair mechanisms. However, a majority of each type had structures consistent with the predictions of our SD-MMEJ model. This suggests that a single underlying mechanism could be responsible for all three repair product types. Genetic analysis indicates that SD-MMEJ is Ku70, Lig4 and Rad51-indepEndent but impaired in mus308 (POLQ) mutants.
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MMEJ repair of double-strand breaks (director’s cut): deleted sequences and alternative Endings
Trends in genetics : TIG, 2008Co-Authors: Mitch Mcvey, Sang Eun LeeAbstract:DNA double-strand breaks are normal consequences of cell division and differentiation and must be repaired faithfully to maintain genome stability. Two mechanistically distinct pathways are known to efficiently repair double-strand breaks: homologous recombination and Ku-depEndent non-homologous End Joining. Recently, a third, less characterized repair mechanism, named Microhomology-Mediated End Joining (MMEJ), has received increasing attention. MMEJ repairs DNA breaks via the use of substantial microhomology and always results in deletions. Furthermore, it probably contributes to oncogenic chromosome rearrangements and genetic variation in humans. Here, we summarize the genetic attributes of MMEJ from several model systems and discuss the relationship between MMEJ and 'alternative End Joining'. We propose a mechanistic model for MMEJ and highlight important questions for future research.
Stephen C Ekker - One of the best experts on this subject based on the ideXlab platform.
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The Gene Sculpt Suite: a set of tools for genome editing.
Nucleic acids research, 2019Co-Authors: Carla M Mann, Hirotaka Ata, Gabriel Martínez-gálvez, Karl J Clark, Jordan M. Welker, Wesley A. Wierson, Maira P. Almeida, Jeffrey J. Essner, Maura Mcgrail, Stephen C EkkerAbstract:The discovery and development of DNA-editing nucleases (Zinc Finger Nucleases, TALENs, CRISPR/Cas systems) has given scientists the ability to precisely engineer or edit genomes as never before. Several different platforms, protocols and vectors for precision genome editing are now available, leading to the development of supporting web-based software. Here we present the Gene Sculpt Suite (GSS), which comprises three tools: (i) GTagHD, which automatically designs and generates oligonucleotides for use with the GeneWeld knock-in protocol; (ii) MEDJED, a machine learning method, which predicts the extent to which a double-stranded DNA break site will utilize the Microhomology-Mediated repair pathway; and (iii) MENTHU, a tool for identifying genomic locations likely to give rise to a single predominant Microhomology-Mediated End Joining allele (PreMA) repair outcome. All tools in the GSS are freely available for download under the GPL v3.0 license and can be run locally on Windows, Mac and Linux systems capable of running R and/or Docker. The GSS is also freely available online at www.genesculpt.org.
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Precision gene editing technology and applications in nephrology
Nature Reviews Nephrology, 2018Co-Authors: Zachary Warejoncas, Gabriel Martínez-gálvez, Jarryd M. Campbell, William A. C. Gendron, Michael A. Barry, Peter C. Harris, Caroline R. Sussman, Stephen C EkkerAbstract:The expanding field of precision gene editing is empowering researchers to directly modify DNA. Gene editing is made possible using synonymous technologies: a DNA-binding platform to molecularly locate user-selected genomic sequences and an associated biochemical activity that serves as a functional editor. The advent of accessible DNA-targeting molecular systems, such as zinc-finger nucleases, transcription activator-like effectors (TALEs) and CRISPR–Cas9 gene editing systems, has unlocked the ability to target nearly any DNA sequence with nucleotide-level precision. Progress has also been made in harnessing Endogenous DNA repair machineries, such as non-homologous End Joining, homology-directed repair and Microhomology-Mediated End Joining, to functionally manipulate genetic sequences. As understanding of how DNA damage results in deletions, insertions and modifications increases, the genome becomes more predictably mutable. DNA-binding platforms such as TALEs and CRISPR can also be used to make locus-specific epigenetic changes and to transcriptionally enhance or suppress genes. Although many challenges remain, the application of precision gene editing technology in the field of nephrology has enabled the generation of new animal models of disease as well as advances in the development of novel therapeutic approaches such as gene therapy and xenotransplantation. Zinc-finger nucleases, transcription activator-like effector nucleases and CRISPR systems are powerful tools that are enabling new applications of genome engineering in diverse systems. Targeted double-stranded breaks in DNA activate diverse repair processes, such as non-homologous End Joining, homology-directed repair and Microhomology-Mediated End Joining, which can be utilized to modify the nucleotide sequence of DNA. Use of non-nuclease genomic tools enables the editing of single bases and locus-specific epigenetic targeting to modify gene expression. Applications of precision gene editing in nephrology include the generation of animal models to investigate kidney development and disease mechanisms as well as the development of targeted gene therapies. Genome editing in the kidney is challenging owing to anatomical barriers to gene delivery, limitations of vector size and immune responses against viral vectors, modified cells and editing proteins. Despite these challenges, precision gene editing has great potential to accelerate basic science in nephrology and to advance clinical practice through the development of novel therapies for renal diseases. The application of precision gene editing has great potential to accelerate basic research and advance clinical practice in nephrology. Here, the authors discuss this technology and the challenges and potential of genome editing in the kidney.
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Toward Precision Molecular Surgery: Robust, Selective Induction of Microhomology-Mediated End Joining in vivo
2018Co-Authors: Hirotaka Ata, Thomas L Ekstrom, Gabriel Martínez-gálvez, Carla M Mann, Alexey V Dvornikov, Kyle J Schaefbauer, Drena Dobbs, Karl J Clark, Alvin C.h., Stephen C EkkerAbstract:One key problem in precision genome editing is the resultant unpredictable plurality of sequence outcomes at the site of targeted DNA double-strand breaks (DSBs). This is due to the typical activation of the versatile Non-homologous End Joining (NHEJ) pathway. Such unpredictability limits the utility of somatic gene editing for applications including gene therapy and functional genomics. For germline editing work, the accurate reproduction of identical alleles using NHEJ is a labor intensive process. In this study, we propose inducing Microhomology-Mediated End Joining (MMEJ) as a viable solution for improving somatic sequence homogeneity in vivo, capable of generating a single predictable allele at high rates (56% ~ 86% of the entire mutant allele pool). Using a combined dataset from zebrafish (Danio rerio) in vivo and human HeLa cell in vitro as a training dataset, we identified specific contextual sequence determinants surrounding genomic DSBs for robust MMEJ pathway activation. We then applied our observation and prospectively designed MMEJ-inducing sgRNAs against a variety of proof-of-principle genes and demonstrated a high level of mutant allele homogeneity at these loci. F0 mutant zebrafish embryos and larvae generated with these gRNAs faithfully recapitulated previously reported, recessive loss-of-function phenotypes. We also provide a novel algorithm MENTHU (http://genesculpt.org/menthu/) for improved prediction of candidate MMEJ loci, suitable for both targeted and genome-wide applications. We believe that this MMEJ-centric approach will have a broad impact on genome engineering and its applications. For example, whereas somatic mosaicism hinders efficient recreation of a knockout mutant allele at base pair resolution via the standard NHEJ-based approach, we demonstrate that F0 founders transmitted the identical MMEJ allele of interest at high rates. Most importantly, the ability to directly dictate the reading frame of an Endogenous target will have important implications for gene therapy applications in human genetic diseases.
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Robust activation of Microhomology-Mediated End Joining for precision gene editing applications.
Public Library of Science (PLoS), 2018Co-Authors: Hirotaka Ata, Thomas L Ekstrom, Gabriel Martínez-gálvez, Carla M Mann, Alexey V Dvornikov, Kyle J Schaefbauer, Alvin C, Drena Dobbs, Karl J Clark, Stephen C EkkerAbstract:One key problem in precision genome editing is the unpredictable plurality of sequence outcomes at the site of targeted DNA double stranded breaks (DSBs). This is due to the typical activation of the versatile Non-homologous End Joining (NHEJ) pathway. Such unpredictability limits the utility of somatic gene editing for applications including gene therapy and functional genomics. For germline editing work, the accurate reproduction of the identical alleles using NHEJ is a labor intensive process. In this study, we propose Microhomology-Mediated End Joining (MMEJ) as a viable solution for improving somatic sequence homogeneity in vivo, capable of generating a single predictable allele at high rates (56% ~ 86% of the entire mutant allele pool). Using a combined dataset from zebrafish (Danio rerio) in vivo and human HeLa cell in vitro, we identified specific contextual sequence determinants surrounding genomic DSBs for robust MMEJ pathway activation. We then applied our observation to prospectively design MMEJ-inducing sgRNAs against a variety of proof-of-principle genes and demonstrated high levels of mutant allele homogeneity. MMEJ-based DNA repair at these target loci successfully generated F0 mutant zebrafish embryos and larvae that faithfully recapitulated previously reported, recessive, loss-of-function phenotypes. We also tested the generalizability of our approach in cultured human cells. Finally, we provide a novel algorithm, MENTHU (http://genesculpt.org/menthu/), for improved and facile prediction of candidate MMEJ loci. We believe that this MMEJ-centric approach will have a broader impact on genome engineering and its applications. For example, whereas somatic mosaicism hinders efficient recreation of knockout mutant allele at base pair resolution via the standard NHEJ-based approach, we demonstrate that F0 founders transmitted the identical MMEJ allele of interest at high rates. Most importantly, the ability to directly dictate the reading frame of an Endogenous target will have important implications for gene therapy applications in human genetic diseases
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Robust activation of Microhomology-Mediated End Joining for precision gene editing applications
'Public Library of Science (PLoS)', 2018Co-Authors: Ata H, Alexey V Dvornikov, Kyle J Schaefbauer, Tl Ekstrom, Martinez-galvez G, Cm Mann, Dobbs D, Kj Clark, Stephen C EkkerAbstract:One key problem in precision genome editing is the unpredictable plurality of sequence outcomes at the site of targeted DNA double stranded breaks (DSBs). This is due to the typical activation of the versatile Non-homologous End Joining (NHEJ) pathway. Such unpredictability limits the utility of somatic gene editing for applications including gene therapy and functional genomics. For germline editing work, the accurate reproduction of the identical alleles using NHEJ is a labor intensive process. In this study, we propose Microhomology-Mediated End Joining (MMEJ) as a viable solution for improving somatic sequence homogeneity in vivo, capable of generating a single predictable allele at high rates (56% similar to 86% of the entire mutant allele pool). Using a combined dataset from zebrafish (Danio rerio) in vivo and human HeLa cell in vitro, we identified specific contextual sequence determinants surrounding genomic DSBs for robust MMEJ pathway activation. We then applied our observation to prospectively design MMEJ-inducing sgRNAs against a variety of proof-of-principle genes and demonstrated high levels of mutant allele homogeneity. MMEJ-based DNA repair at these target loci successfully generated FO mutant zebrafish embryos and larvae that faithfully recapitulated previously reported, recessive, loss-of-function phenotypes. We also tested the generalizability of our approach in cultured human cells. Finally, we provide a novel algorithm, MENTHU (http://genesculpt.org/menthu/), for improved and facile prediction of candidate MMEJ loci. We believe that this MMEJ-centric approach will have a broader impact on genome engineering and its applications. For example, whereas somatic mosaicism hinders efficient recreation of knockout mutant allele at base pair resolution via the standard NHEJ-based approach, we demonstrate that F0 founders transmitted the identical MMEJ allele of interest at high rates. Most importantly, the ability to directly dictate the reading frame of an Endogenous target will have important implications for gene therapy applications in human genetic diseases.Department of Health Technology and Informatics201901 bcrcpublished_fina
Tetsushi Sakuma - One of the best experts on this subject based on the ideXlab platform.
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Biased genome editing using the local accumulation of DSB repair molecules system
Nature communications, 2018Co-Authors: Shota Nakade, Tomomi Aida, Tetsushi Sakuma, Keiji Mochida, Kohichi Tanaka, Naoaki Sakamoto, Atsushi Kunii, Kazuki Nakamae, Takashi YamamotoAbstract:Selective genome editing such as gene knock-in has recently been achieved by administration of chemical enhancer or inhibitor of particular DNA double-strand break (DSB) repair pathways, as well as overexpression of pathway-specific genes. In this study, we attempt to enhance the efficiency further to secure robust gene knock-ins, by using the local accumulation of DSB repair molecules (LoAD) system. We identify CtIP as a strong enhancer of Microhomology-Mediated End-Joining (MMEJ) repair by genetic screening, and show the knock-in-enhancing effect of CtIP LoADing. Next-generation sequencing reveals that CtIP LoADing highly increases the frequency of MMEJ-mediated integration. Selection-free, simultaneous triple gene knock-ins are also achieved with the CtIP-LoADing strategy. Moreover, by replacing the LoADing molecules and targeting strategies, this system can be applied for other specific genome engineering purposes, such as introducing longer deletions for gene disruption, indepEndently introducing multiple mutations without chromosomal deletion, and efficiently incorporating a single-stranded oligodeoxynucleotide donor. Genome editing using CRISPR can be enhanced by manipulating DNA double-strand break repair pathways. Here the authors demonstrate LoAD, local accumulation of repair molecules, which shifts repair to Microhomology-Mediated End-Joining.
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Microhomology-assisted scarless genome editing in human iPSCs
Nature communications, 2018Co-Authors: Shin Il Kim, Tetsushi Sakuma, Tomoko Matsumoto, Harunobu Kagawa, Michiko Nakamura, Ryoko Hirohata, Ayano Ueno, Maki Ohishi, Tomoyoshi Soga, Takashi YamamotoAbstract:Gene-edited induced pluripotent stem cells (iPSCs) provide relevant isogenic human disease models in patient-specific or healthy genetic backgrounds. Towards this End, gene targeting using antibiotic selection along with engineered point mutations remains a reliable method to enrich edited cells. Nevertheless, integrated selection markers obstruct scarless transgene-free gene editing. Here, we present a method for scarless selection marker excision using engineered Microhomology-Mediated End Joining (MMEJ). By overlapping the homology arms of standard donor vectors, short tandem microhomologies are generated flanking the selection marker. Unique CRISPR-Cas9 protospacer sequences nested between the selection marker and engineered microhomologies are cleaved after gene targeting, engaging MMEJ and scarless excision. Moreover, when point mutations are positioned unilaterally within engineered microhomologies, both mutant and normal isogenic clones are derived simultaneously. The utility and fidelity of our method is demonstrated in human iPSCs by editing the X-linked HPRT1 locus and biallelic modification of the autosomal APRT locus, eliciting disease-relevant metabolic phenotypes.
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Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ
BMC Genomics, 2016Co-Authors: Tomomi Aida, Shota Nakade, Yayoi Izu, Ayu Oishi, Hidenori Aizawa, Tetsushi Sakuma, Harumi Ishikubo, Keiji Mochida, Takako Usami, Takashi YamamotoAbstract:Although CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by Microhomology-Mediated End-Joining. Here, we identified exonuclease 1 (Exo1) as an enhancer for PITCh in human cells. By combining the Exo1 and PITCh-directed donor vectors, we achieved convenient one-step knock-in of gene cassettes and floxed allele both in human cells and mouse zygotes. Our results provide a technical platform for high-throughput knock-in.
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Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ
BMC Genomics, 2016Co-Authors: Tomomi Aida, Shota Nakade, Ayu Oishi, Hidenori Aizawa, Tetsushi Sakuma, Harumi Ishikubo, Keiji Mochida, Takako Usami, Takashi Yamamoto, Kohichi TanakaAbstract:Background Although CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by Microhomology-Mediated End-Joining. Results Here, we identified exonuclease 1 ( Exo1 ) as an enhancer for PITCh in human cells. By combining the Exo1 and PITCh-directed donor vectors, we achieved convenient one-step knock-in of gene cassettes and floxed allele both in human cells and mouse zygotes. Conclusions Our results provide a technical platform for high-throughput knock-in.
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Microhomology-Mediated End-Joining-depEndent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9.
Nature communications, 2014Co-Authors: Shota Nakade, Takashi Yamamoto, Takuya Tsubota, Yuto Sakane, Satoshi Kume, Naoaki Sakamoto, Masanobu Obara, Takaaki Daimon, Hideki Sezutsu, Tetsushi SakumaAbstract:One challenge facing the use of programmable nucleases in genome engineering is the requirement for homologous recombination. Here, Nakade et al. harness Microhomology-Mediated End-Joining as a means of inserting exogenous coding sequences into the genome using both TALEN and CRISPR/Cas9 technologies.