Chromatin structure of two genomic sites for targeted transgene integration in induced pluripotent stem cells and hematopoietic stem cells

Rensburg, Ruan van; Beyer, Ines; Yao, Xiaoying; Wang, H.; Denisenko, Oleg; Li, Z. Y.; Russell, David W.; Miller, Daniel G.; Gregory, Philip D.; Holmes, Michael C.; Bomsztyk, Karol; Lieber, André

doi:10.1038/gt.2012.25

Cited by 40 publications

(25 citation statements)

References 68 publications

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“…Epigenetic status is known to affect all DNA-metabolism processes including transcription, replication, and repair (Cavalli and Misteli, 2013; Papamichos-Chronakis and Peterson, 2013). The importance of transcriptional activation and epigenetic status for gene editing efficiency is still largely unknown, but epigenetic modification was recently shown to impact DSBR pathway choice (Daboussi et al, 2012; Kuhar et al, 2014; Valton et al, 2012a; Valton et al, 2012b; van Rensburg et al, 2013). The SMRT DNA sequencing strategy could be used to further study how chromatin status influences DSBR pathway choice and gene editing efficiency by providing analysis in a broader range of cell types in which the chromatin state of the targeted site is known.…”

Section: Discussionmentioning

confidence: 99%

Quantifying Genome-Editing Outcomes at Endogenous Loci with SMRT Sequencing

et al. 2014

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SUMMARY Targeted genome editing with engineered nucleases has transformed the ability to introduce precise sequence modifications at almost any site within the genome. A major obstacle to probing the efficiency and consequences of genome editing is that no existing method enables the frequency of different editing events to be simultaneously measured across a cell population at any endogenous genomic locus. We have developed a novel method for quantifying individual genome editing outcomes at any site of interest using single molecule real time (SMRT) DNA sequencing. We show that this approach can be applied at various loci, using multiple engineered nuclease platforms including TALENs, RNA guided endonucleases (CRISPR/Cas9), and ZFNs, and in different cell lines to identify conditions and strategies in which the desired engineering outcome has occurred. This approach facilitates the evaluation of new gene editing technologies and permits sensitive quantification of editing outcomes in almost every experimental system used.

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Section: Discussionmentioning

confidence: 99%

Quantifying Genome-Editing Outcomes at Endogenous Loci with SMRT Sequencing

et al. 2014

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“…Finally, although the efficiency of ZFR-mediated integration is lower than that achieved by zinc-finger (43,44) or TAL effector (22) nuclease-based approaches, we anticipate that optimization of the ZFR architecture will lead to reduced off-target integration events and higher targeting efficiency. Additional studies aimed at evaluating whether ZFR activity is cell type (25) or chromatin structure dependent (45) may also help establish limitations and clarify opportunities for ZFR targeting. In conclusion, we have developed a diverse collection of re-engineered Gin recombinase catalytic domains suitable for the design of ZFRs with custom specificity.…”

Section: Discussionmentioning

confidence: 99%

A comprehensive approach to zinc-finger recombinase customization enables genomic targeting in human cells

Gaj

Mercer

Sirk

et al. 2013

Nucleic Acids Research

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Zinc-finger recombinases (ZFRs) represent a potentially powerful class of tools for targeted genetic engineering. These chimeric enzymes are composed of an activated catalytic domain derived from the resolvase/invertase family of serine recombinases and a custom-designed zinc-finger DNA-binding domain. The use of ZFRs, however, has been restricted by sequence requirements imposed by the recombinase catalytic domain. Here, we combine substrate specificity analysis and directed evolution to develop a diverse collection of Gin recombinase catalytic domains capable of recognizing an estimated 3.77 × 107 unique DNA sequences. We show that ZFRs assembled from these engineered catalytic domains recombine user-defined DNA targets with high specificity, and that designed ZFRs integrate DNA into targeted endogenous loci in human cells. This study demonstrates the feasibility of generating customized ZFRs and the potential of ZFR technology for a diverse range of applications, including genome engineering, synthetic biology and gene therapy.

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“…Thus, insertion of exogenous DNA into the human AAVS1 locus by genome editing increases cellular contractile force even though fundamental cellular features such as cell morphology and cell growth are maintained. Previous research showed that transgene integration into the human AAVS1 locus did not change cell growth rate [13] or pluripotency [2]. Thus, the AAVS1 locus is thought to be a strong candidate for gene therapy.…”

Section: Reduction Of Mbs85 Expression Is Associated With Myosin Regumentioning

confidence: 99%

“…Although no genomic site has been fully validated as a genomic "safe harbor", some candidates from the human genome have been proposed, including the adeno-associated virus site 1 (AAVS1) locus, the chemokine (CC motif) receptor 5 (CCR5) locus, and the human orthologue of the mouse ROSA26 locus [1]. Among these candidates, the AAVS1 locus stands out for the ubiquitous, constitutive, and high-level expression of the integrated transgene [2]. Mali et al proposed a revolutionary method for the insertion of transgenes into the AAVS1 locus with the development of CRISPR/Cas system [3].…”

Section: Introductionmentioning

confidence: 99%

Transgene integration into the human AAVS1 locus enhances myosin II-dependent contractile force by reducing expression of myosin binding subunit 85

Mizutani

Haga

et al. 2015

Biochemical and Biophysical Research Communications

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The adeno-associated virus site 1 (AAVS1) locus in the human genome is a strong candidate for gene therapy by insertion of an exogenous gene into the locus. The AAVS1 locus includes the coding region for myosin binding subunit 85 (MBS85). Although the function of MBS85 is not well understood, myosin II-dependent contractile force may be affected by altered expression of MBS85. The effect of altered expression of MBS85 on cellular contractile force should be examined prior to the application of gene therapy. In this study, we show that transgene integration into AAVS1 and consequent reduction of MBS85 expression changes myosin II-dependent cellular contractile force. We established a human fibroblast cell line with exogenous DNA knocked-in to AAVS1 (KI cells) using the CRISPR/Cas9 genome editing system. Western blotting analysis showed that KI cells had significantly reduced MBS85 expression. KI cells also showed greater cellular contractile force than control cells. The increased contractile force was associated with phosphorylation of the myosin II regulatory light chain (MRLC). Transfection of KI cells with an MBS85 expression plasmid restored cellular contractile force and phosphorylation of MRLC to the levels in control cells. These data suggest that transgene integration into the human AAVS1 locus induces an increase in cellular contractile force and thus should be considered as a gene therapy to effect changes in cellular contractile force. 3 Highlights•Transgene integration to human AAVS1 locus decreases MBS85 expression.•Decrease in MBS85 expression increases cellular contractile force.•MBS85 is involved in myosin regulatory light chain phosphorylation.

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Chromatin structure of two genomic sites for targeted transgene integration in induced pluripotent stem cells and hematopoietic stem cells

Cited by 40 publications

References 68 publications

Quantifying Genome-Editing Outcomes at Endogenous Loci with SMRT Sequencing

Quantifying Genome-Editing Outcomes at Endogenous Loci with SMRT Sequencing

A comprehensive approach to zinc-finger recombinase customization enables genomic targeting in human cells

Transgene integration into the human AAVS1 locus enhances myosin II-dependent contractile force by reducing expression of myosin binding subunit 85

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