Methods xxx (2014) xxx–xxx
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TAL effector-mediated genome visualization (TGV) Yusuke Miyanari Okazaki Institute for Integrative Bioscience, Okazaki, Japan
a r t i c l e
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Article history: Received 2 December 2013 Revised 6 March 2014 Accepted 25 March 2014 Available online xxxx Keywords: TAL effector Nuclear architecture Live imaging
a b s t r a c t The three-dimensional remodeling of chromatin within nucleus is being recognized as determinant for genome regulation. Recent technological advances in live imaging of chromosome loci begun to explore the biological roles of the movement of the chromatin within the nucleus. To facilitate better understanding of the functional relevance and mechanisms regulating genome architecture, we applied transcription activator-like effector (TALE) technology to visualize endogenous repetitive genomic sequences in mouse cells. The application, called TAL effector-mediated genome visualization (TGV), allows us to label specific repetitive sequences and trace nuclear remodeling in living cells. Using this system, parental origin of chromosomes was specifically traced by distinction of single-nucleotide polymorphisms (SNPs). This review will present our approaches to monitor nuclear dynamics of target sequences and highlights key properties and potential uses of TGV. Ó 2014 Published by Elsevier Inc.
1. Introduction 1.1. Nuclear architecture DNA is packaged by forming nucleosomes, called chromatin. Chromatin fibers are organized non-randomly within the nuclear space and move continually even in interphase, not only due to temperature-dependent Brownian motion [1,2] but also involving active movement [3–5]. Spatiotemporal organization of chromatin within nucleus is suggested as an emerging key player to regulate genome functions including gene expression [6]. The developmental program which requires a precise control of gene expression accompanies nuclear remodeling, resulting in the generation of cell-type specific nuclear architecture. Moreover, defects in nuclear reorganization lead to developmental aberrations and several human diseases [7]. Despite the drastic change of nuclear structure during cell differentiation, its functional role in cell-fate decision remains largely unexplored.
target genomic regions in living cells, where integration of exogenous array of LacO DNA binding sequences into the genome is required [8]. In this system, LacI fused with monomeric fluorescent protein specifically binds to LacO array, resulting in detection of subnuclear positioning of the target region as a visible fluorescent spot. However, since introduction of the large exogenous LacO array (10 kb) into the genome usually occurs at random positions, the technique is in fact inapplicable for in vivo imaging of specific endogenous genomic loci. In 2007, Lindhout et al. reported a new approach for live visualization of endogenous genomic sequences using zinc finger-DNA recognition codes [9]. Expression of zinc finger proteins fused with GFP, which are designed to bind specific repetitive DNA sequences, allows imaging of subnuclear localization of the target sequences in living cells. Based on this concept, we developed TAL effector-mediated genome visualization (TGV), which offers several advantages including ease of design, its potential ability to be applied to any sequences and simpler optimization [10]. 1.3. TAL effector
1.2. Applications to study nuclear organization DNA fluorescent in situ hybridization (DNA-FISH) is currently the method of choice to study subnuclear positioning of target sequences with microscopy. Since it is performed with fixed cells, live imaging of nuclear remodeling cannot be addressed by DNAFISH. Fluorescent Lac repressor (LacI) system allows imaging of E-mail address:
[email protected] TALEs, proteins discovered in the plant pathogenic bacteria Xanthomonas, have emerged as powerful scaffolds for engineering DNA binding proteins [11,12]. TALE proteins are composed of tandem repeat of 34 amino acids (TALE repeats) (Fig. 1A). Their sequences are nearly identical between these repeats except for two variable amino acids, referred as repeat-variable diresidue (RVDs), that define the base-recognition specificity of each unit [11,12]. Therefore arrays of four different repeat units allow us to
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Fig. 1. (A) Schematic diagram of a fluorescent TALE and target sequence. Truncated TALE (N153AA and C63AA) was fused with nuclear localization signal (NLS) at N-terminus and monomeric fluorescent protein such as mClover at C-terminus. Central domain of TALE is composed of tandem array of 34 aa TALE repeat harboring repeat-variable diresidue (RVDs). TALE code representing specificity of RVDs to corresponding nucleotide is shown on the right side. (B) Work flow for selection of fluorescent TALEs for TGV.
generate TALEs with user-defined specificity. Using this simple code, TALE is a versatile platform for engineering DNA-binding proteins with specific functionality upon fusion with operative proteins such as nucleases [13], transcriptional modulators [14–16], recombinases [17], and epigenetic modifiers [18,19]. Here I will highlight the application that we developed, and that we refer to as TGV, using TALEs fused with fluorescent proteins to visualize the subnuclear positioning of repetitive sequences in living cells [10]. I also discuss the potential target sequences of TGV and alternative approaches to visualize repetitive sequences and unique loci.
2. Materials and methods 2.1. Design of TALEs for TGV 2.1.1. Identification of target sequences Target DNA sequences of TALEs were identified from sequences of interest using the TAL effector Nucleotide Targeter (TALE-NT) 2.0
website (https://tale-nt.cac.cornell.edu/about) (Fig. 1B) [20,21]. Sequences of 15 nt or longer are appropriate for specific labeling of target sequences, since TALE against shorter sequence displayed higher background signal in the nucleoplasm as compared with TALE recognizing 15 nt or longer, possibly due to lower binding affinity or lower specificity to the target sequences [10]. It should be noted that DNA methylation on target sequences could affect binding affinity of TALEs, since TALE repeat with the HD RVD is sensitive to methylation at cytosine [22]. Hence, it is safer to select DNA sequences without CpG sites which are potentially methylated. Alternatively, N⁄ RVD repeats could be used for recognizing methylated cytosine [22]. NH RVD module was used for recognizing guanine, since it displays higher specificity than substituting NN RVD module, which has affinity for both guanine and adenine [23,24].
2.1.2. Structure of fluorescent TALEs TALEs for TGV were fused with nuclear localization signal (NLS) at N-terminus and fluorescent protein at C-terminus [10] (Fig. 1A).
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Some fluorescent proteins such as EGFP indeed display weak dimmerization activity [25], which could potentially have abnormal effects to its fusion proteins. To avoid any effect from fluorescent proteins, monomeric fluorescent proteins such as mClover and mRuby2 are applicable for TGV.
(available from Addgene) composed of mClover, mRuby2, VP64 transcription activation domain, and 3xTy1 tags, respectively, were used as destination plasmids.
2.2. Assembly of TALEs
TALEs fused with VP64 transcription activation domain were constructed as described above with pcDNA-TALvp64 (available from Addgene). HEK293T cells cultured onto CellBIND 96 well plates (CLS3340-50EA, Sigma) were transfected with 50 ng of Firefly reporter plasmids containing the target sequences of TALEs and minimal CMV promoter, 100 ng of TALE-Vp64 expression plasmids, and 1 ng of pRL-CMV using FuGENE 6 (E2691, Promega). pRL-CMV (Promega), a Renilla reporter plasmid, was used to normalize Firefly luciferase values for transfection efficiency in each well. Firefly and Renilla luciferase activities were measured 48 h after transfection with Dual-Luciferase Reporter Assay System (E1910, Promega).
TALEs were assembled by the Golden Gate approach (Golden Gate TALEN and TAL Effector Kit 2.0 available from Addgene) as described previously [20] with slight modifications (Fig. 2). Briefly, all the plasmids required for the first golden gate reaction, containing each RVD module and pFUS_A or pFUS_B (25 lg each) were mixed in a 200 ll PCR tube, and then concentrated by ethanol precipitation to a final volume of 3 ll of H2O. The following reagents were added to the tube: 0.6 ll of 10 T4 DNA ligase buffer (M0202M, NEB), 0.6 ll of 10 BSA (NEB), 0.3 ll of 10 U/ll BsaI, 0.3 ll of T4 DNA Ligase (M0202M, NEB), and 1.2 ll of H2O. The reaction was incubated in a thermocycler for 40 cycles of 5 min at 37 °C and 10 min at 16 °C, then heated to 50 °C for 5 min and 80 °C for 5 min. During the reaction, RVD region on each plasmid flanked by BsaI site is cleaved out at 37 °C and then ligated with neighboring RVD regions in a proper alignment at 16 °C. Then, 0.3 ll of 25 mM ATP and 0.3 ll of Plasmid Safe DNase (E3105K, Epicentre), which digests linear DNA including partial arrays, are added to the reaction. The mixture was incubated at 37 °C for 30 min, then used for transformation into Escherichia coli SURE2 (200152, Agilent) cells. For the second golden gate reaction, 6 cycles of thermal reaction were performed with 4 ll of reaction mixture as in the first reaction but with 20 ng each of assembled pFUS_A, pFUS_B, pLR, and destination plasmid, and BsmBI instead of BsaI. pTALYM3, pTALYM4, pcDNA-TALvp64, and pTALYM9
2.3. Luciferase assay
2.4. DNA-FISH ES cells expressing fluorescent TALEs were seeded into a well of micro-insert 4 well (80409, Ibidi) placed on a glass bottom dish coated with Laminin-511 (LN511, BioLamina). After fixation with 4% formaldehyde/1PBS for 10 min, cells were stained with 4,6-diamidino-2-phenylindole (DAPI). Images of TGV were acquired with an inverted TCS SP5 Leica confocal microscope. The cells were then subjected to DNA-FISH as described previously [26]. A PCR product was used as a probe for major satellite detection. The primers used to generate the major satellite probe were: forward major satellite primer: 50 -GCGAGAAAACTGAAAATCAC-30 , and reverse major satellite: 50 -TCAAGTCGTCAAGTGGATG-30 . Mouse genomic
Fig. 2. Schematic diagram of TALE assembly by golden gate reaction. To assemble TALE against 13 nucleotide, RVDs for initial 10 nucleotides and remaining 3 nucleotides are separately assembled by 1st golden gate reaction (GGR) into pFUSA and pFUSB plasmid, respectively. Resulting pFUSA-10 and pFUSB-13 are concatenated with a destination plasmid pTALYM3 harboring ampicillin resistant cassette (Ampr), T7 promoter, chicken beta-actin (CAG) promoter, N- and C-terminus of TALE, mClover, and poly A sequence by 2nd golden gate reaction. Final construct is applicable for both in vitro transcription and ectopic expression in mammalian cells.
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DNA was used as a template for PCR reaction. Handmade Atto647NdATP was included in the PCR reaction at a concentration of 20 lM to generate the fluorescent probes. The identical fields of acquisition were acquired to overlay images of TGV with corresponding DNA-FISH signals. 2.5. Chromatin immunoprecipitation (ChIp) assay To verify binding specificity of TALEs to target sequences, ChIP assay was performed with ES cells expressing TALE-Ty1 against major satellite sequence as previously described [27]. 1 lg of anti-Ty1 antibody (Mab-054-050, Diagenode) was used for the assay. The enrichment of TALE-Ty1 was quantified by real-time PCR (LightCycler 480, Roche) and normalized to input DNA. Primers used in this assay were described previously [10]. 2.6. Live cell imaging Cells were grown on culture inserts placed on 3-cm glass-bottomed dishes (81158, Ibidi) coated with Laminin-511. Each dish was placed in an incubation chamber (Tokai Hit) on the microscope stage at 37 °C under a 5% CO2 atmosphere. An inverted confocal microscope (Leica) attached to a Nipkow disk (CSU22, Yokogawa Electric) with an EMCCD camera (iXon, Andor Technology) was controlled with iQ software (Andor Technology). mClover, mRuby2, and Histone H2B-tdiRFP were excited with 488-nm, 560-nm and 640-nm lasers, respectively. Images for each color (green or red) were acquired across 15 mm (16 Z-planes) every 5–10 min for 12 h.
expressed it in mouse embryonic stem (ES) cells. Fluorescent signals of mClover were predominantly detected at pericentromeric regions (Fig. 3B), suggesting that the TALE-mClover specifically binds to major satellite sequences with no signs of cytotoxicity or abnormal nuclear morphology. We indeed observed aggregation of major satellite domains resulting in formation of large fluorescent loci in ES cells expressing the TALE fused with conventional EGFP (data not shown). This might be due to weak dimerization activity of EGFP [25], since we did not observed abnormal morphology with TALE fused with mClover, which harbors a monomeric point mutation A206K. Therefore, monomeric fluorescent protein is applicable for TGV to avoid abnormal chromatin structure on target sequences. We found that the pericentromeric regions were efficiently labeled throughout cell-cycle in both cultured ES cells and mouse preimplantation embryos (Fig. 3C). Importantly, expression of the TALE did not exhibit detectable cytotoxicity. The flexible nature in the design of TALEs allowed us to visualise other repetitive sequences including centromeric minor satellite (MinSat) and telomeric repeats [10], suggesting that TGV system can be potentially applied to other repetitive sequences. It is recommended to construct several TALEs for each target sequence because not every TALE works as expected. For example, TALE against telomeric G-strand sequence (50 -TAGGGTTAGGGTTAGG-30 ) but not C-strand sequence (50 -TAAC CCTAACCCTAAC-30 ) binds to telomere (data not shown). This might be due to low binding affinity of the latter TALE or competition with endogenous proteins on the C-strand. 3.2. Differential labeling of parental chromosome by distinction of SNPs
3. Imaging of repetitive sequences 3.1. Detection of endogenous repetitive sequences We designed TALE to recognize major satellite sequences which are highly abundant tandem repeats in pericentromeric regions of all mouse chromosome, except Y chromosome (Fig. 3A). We fused the TALE with monomeric green fluorescent protein, mClover, and
One of the great features of TALEs is that their binding specificity completely depends on target sequences, which potentially allows us to distinguish chromosomes in a parent-of-origin manner in living cells by recognition of SNPs. To test this possibility, we used hybrid ES cells (SF1) derived from F1 embryos between Mus musculus and Mus spretus [28] because these two species have different sequences in satellite repeats [29]. Moreover, the abundance of
Fig. 3. Specific labeling of major satellite sequence by TGV in living cells. Schematic diagram of mouse chromosome. The location of telomeres (blue), pericentoromeric major satellites (green), centromeric minor satellites (red), and the long arm of the chromosome (gray) are indicated. (A) Fluorescent images of mitotic chromosomes of ES cells expressing TALE-mClover against 15 nt of major satellite sequence. Images for mClover (green) and DAPI (magenta) are shown with merged image. (B) Representative images of mouse preimplantation embryos expressing TALE-mClover_MajSat (green) and H2B-mRFP (magenta) at the indicated developmental stages. Dashed lines indicate the nuclear membrane. Scale bar, 20 lm. Panel A and C were reprinted from Nature Structural & Molecular Biology, vol. 20, No. 11, Miyanari et al. ‘‘Live visualization of chromatin dynamics with fluorescent TALEs’’, p1321–1324, Copyright (2013).
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Fig. 4. Differential labeling of parental chromosome by distinction of SNPs. (A) Schematic of M. musculus and M. spretus chromosomes. M. spretus chromosomes consist of large domains of minor satellites and small domains of major satellites, in contrast to M. musculus chromosomes. (B) Sequence alignment of minor satellite from M. musculus and M. spretus. Asterisks, positions of SNPs; arrow, target sequence used for the parental M. spretus TALE-mRuby2. (C) Time-lapse snapshots from live imaging of SF1 cells transiently expressing the TALE-mClover_MajSat (green), TALE-mRuby2_ MinSat (magenta) and tandem infrared fluorescent protein–tagged H2B (H2B-tdiRFP) (gray). Scale bars, 1 lm. All the panels were reprinted from Nature Structural & Molecular Biology, vol. 20, No. 11, Miyanari et al. ‘‘Live visualization of chromatin dynamics with fluorescent TALEs’’, p1321–1324, Copyright (2013).
Fig. 5. Characterization of TALEs. (A) Schematic diagram of reporter assay to characterize binding affinity and specificity of TALEs. 293T cells are is transfected with a plasmid encoding TALE fused with VP64, a transcription activating domain, and a reporter plasmid containing tandem repeats of the binding sequence upstream of a minimal CMV promoter. (B) DNA-FISH with a major-satellite probe on ES cells expressing TALE-mClover_MajSat. Images for mClover (green, left) and DNA-FISH (magenta, right) are shown together with DAPI staining (gray). (C) Anti-Ty1 ChIP of the TALE-Ty1_MajSat on the indicated repetitive sequences in ES cells. Control, nontransfected cells. The mean ± s.d. of three independent biological replicates is shown. Statistical comparisons were performed by two-tailed Student’s t test. Panel B and C were reprinted from Nature Structural & Molecular Biology, vol. 20, No. 11, Miyanari et al. ‘‘Live visualization of chromatin dynamics with fluorescent TALEs’’, p1321–1324, Copyright (2013).
MajSat and MinSat sequences between the two species is different [30]. M. spretus chromosomes harbor a higher copy number of MinSat and fewer copies of MajSat in comparison with M. musculus chromosomes (Fig. 4A). To distinguish maternal and paternal chromosomes by different colors, we targeted TALE-mClover against MajSat and TALE-mRuby2 against MinSat, respectively. For specific labeling of chromosomes of maternal origin, TALE-mRuby2 was designed to specifically recognize the MinSat sequence of M. spretus, containing two SNPs compared with that of M. musculus (Fig. 4B). These two fluorescent TALEs allowed us to specifically distinguish pericentromeric regions of maternal and paternal chromosomes
throughout all stages of cell-cycle in SF1 cells (Fig. 4C). We also confirmed that even one SNP within a 15-nt target sequence efficiently discriminates between parental alleles [10]. This result confirms high specificity of TALEs and also shows TALE as a powerful tool for allele-specific labeling by distinction of SNPs in living cells.
4. Characterization of TALEs It should be noted that that characterization of TALEs to understand their binding affinity, specificity, and side effects should be
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carefully performed for each TALE construct (Fig. 1B), since all these features depends on the target sequences of TALEs. 4.1. Evaluation of binding affinity and specificity of TALEs Binding affinity and specificity of TALEs are predictable by luciferase-based reporter assay, where relative luciferase activities driven by binding of TALEs fused with VP64 transcription activator domain to their target sequences on the reporter plasmid are measured (Fig. 5A). Validation of binding specificity to endogenous target sequences is performed by comparison between fluorescent signals from TGV and DNA-FISH. In the case of TALE against major satellite sequence, we confirmed that the fluorescent foci of the TALE-mClover colocalized with those of DNA-FISH for MajSat repeats (Fig. 5B), demonstrating that the TALE specifically binds endogenous MajSat repeats. It is also important to check the efficient binding of TALEs to the target sequences throughout all the stages of cell-cycle by acquiring cells at each cell-cycle. Moreover, we confirmed the specificity of TALE binding by performing chromatin immunoprecipitation (ChIP) assay with ES cells expressing TALE fused with triple Ty1 tags (Fig. 5C). The MajSat sequence was significantly enriched in the material immunoprecipitated with the anti-Ty1 antibody, in contrast to less enrichment of other repetitive sequences including MinSat repeats, long interspersed nuclear elements (LINE1), short interspersed nuclear elements (SINE) B2 or mouse intracisternal type A particle (IAP). Binding of TALEs to putative off-target sites which have similar sequences to the targets can be also evaluated by ChIp-Seq assay. 4.2. Validation of side effects It is important to keep in mind that binding of TALEs to target sites could affect their genome functions through competition with endogenous chromatin-associated proteins such as regulatory transcription factors and replication machinary, or inducing abnormal chromatin configurations including histone occupancy and epigenetic modifications. We confirmed that expression of TALEs against major satellite had no evident side-effect on cell proliferation, chromosome segregation, and chromatin configuration [10]. In fact, we observed a shortened telomere length in cells expressing high levels of TALE against telomere as compared to clones expressing lower levels of TALE, suggesting that higher expression of TALE might have some detrimental effects on chromatin function [10]. This suggests that it is crucial to optimize expression level of TALEs, which dose not affect genome function. 5. Conclusion and discussion It is well established that chromatin configurations play fundamentally important roles in genome function including transcriptional regulation. The functional properties of genomes, however, are determined by more complex mechanisms. The spatial and temporal organization of genomes within 3D space of nucleus is an key player in genome functions, yet still poorly understood. Live imaging of the nuclear organization would be a breakthrough towards uncovering the functional relevance between nuclear architecture and genome functions. TALEs provide a flexible tool to visualize chromatin dynamics. TGV approach allows monitoring of the spatiotemporal organization of repetitive target sequences in cultured cells and living organisms. Live imaging of satellite sequences could be useful to study dynamics of heterochromatin formation during mouse development. High specificity of TALEs also allow us to differentially label parental origin of chromosomes by distinction of SNPs. We have succeeded to visualize a few repetitive sequences including mouse major satellite, minor satellite,
and telomere sequences. Flexibility of TALE design can potentially allows detection of other repetitive sequences such as long interspersed nuclear elements (LINE), short interspersed nuclear elements (SINE), and Long terminal repeats (LTRs) retrotransposon. Whereas this application is limited to repetitive sequences so far, further improvements with an adequate number of targeted TALEs will be necessary to achieve imaging of single-copy loci. RNAguided genome engineering based on CRISPR/Cas system provides an efficient and versatile method for targeted manipulation of mammalian genomes [31]. The binding specificity of CRISPR/Cas protein is easily programmed by changing guide RNA sequences, which is more user-friendly than constructing TALE proteins. Catalytic inactive Cas9 fused with fluorescent protein could be potentially integrated into visualization of target chromatin instead of TALEs. Acknowledgements I appreciate Maria-Elena Torres-Padilla (IGBMC) for crucial reading of the manuscript. This work was supported by an European Molecular Biology Organization (EMBO) long-term fellowship (ALTF864-2008, 2009) and a Japan Society for the Promotion of Science postdoctoral fellowship (2010–2011). References [1] W.F. Marshall, A. Straight, J.F. Marko, J. Swedlow, A. Dernburg, A. Belmont, A.W. Murray, D.A. Agard, J.W. Sedat, Curr. Biol. 7 (1997) 930–939. [2] S. Hihara, C.G. Pack, K. Kaizu, T. Tani, T. Hanafusa, T. Nozaki, S. Takemoto, T. Yoshimi, H. Yokota, N. Imamoto, Y. Sako, M. Kinjo, K. Takahashi, T. Nagai, K. Maeshima, Cell Rep. 2 (2012) 1645–1656. [3] H. Bornfleth, P. Edelmann, D. Zink, T. Cremer, C. Cremer, Biophys. J. 77 (1999) 2871–2886. [4] F.R. Neumann, V. Dion, L.R. Gehlen, M. Tsai-Pflugfelder, R. Schmid, A. Taddei, S.M. Gasser, Genes Dev. 26 (2012) 369–383. [5] S.C. Weber, A.J. Spakowitz, J.A. Theriot, Proc. Natl. Acad. Sci. USA 109 (2012) 7338–7343. [6] J.R. Dixon, S. Selvaraj, F. Yue, A. Kim, Y. Li, Y. Shen, M. Hu, J.S. Liu, B. Ren, Nature 485 (2012) 376–380. [7] T. Misteli, Cold Spring Harb. Perspect. Biol. 2 (2010) a000794. [8] C.C. Robinett, A. Straight, G. Li, C. Willhelm, G. Sudlow, A. Murray, A.S. Belmont, J. Cell Biol. 135 (1996) 1685–1700. [9] B.I. Lindhout, P. Fransz, F. Tessadori, T. Meckel, P.J. Hooykaas, B.J. van der Zaal, Nucleic Acids Res. 35 (2007) e107. [10] Y. Miyanari, C. Ziegler-Birling, M.E. Torres-Padilla, Nat. Struct. Mol. Biol. 20 (2013) 1321–1324. [11] J. Boch, H. Scholze, S. Schornack, A. Landgraf, S. Hahn, S. Kay, T. Lahaye, A. Nickstadt, U. Bonas, Science 326 (2009) 1509–1512. [12] M.J. Moscou, A.J. Bogdanove, Science 326 (2009) 1501. [13] N. Sun, H. Zhao, Biotechnol. Bioeng. 110 (2013) 1811–1821. [14] F. Zhang, L. Cong, S. Lodato, S. Kosuri, G.M. Church, P. Arlotta, Nat. Biotechnol. 29 (2011) 149–153. [15] P. Perez-Pinera, D.G. Ousterout, J.M. Brunger, A.M. Farin, K.A. Glass, F. Guilak, G.E. Crawford, A.J. Hartemink, C.A. Gersbach, Nat. Methods 10 (2013) 239–242. [16] M.L. Maeder, S.J. Linder, D. Reyon, J.F. Angstman, Y. Fu, J.D. Sander, J.K. Joung, Nat. Methods 10 (2013) 243–245. [17] A.C. Mercer, T. Gaj, R.P. Fuller, C.F. Barbas 3rd, Nucleic Acids Res. 40 (2012) 11163–11172. [18] E.M. Mendenhall, K.E. Williamson, D. Reyon, J.Y. Zou, O. Ram, J.K. Joung, B.E. Bernstein, Nat. Biotechnol. 31 (2013) 1133–1136. [19] M.L. Maeder, J.F. Angstman, M.E. Richardson, S.J. Linder, V.M. Cascio, S.Q. Tsai, Q.H. Ho, J.D. Sander, D. Reyon, B.E. Bernstein, J.F. Costello, M.F. Wilkinson, J.K. Joung, Nat. Biotechnol. 31 (2013) 1137–1142. [20] T. Cermak, E.L. Doyle, M. Christian, L. Wang, Y. Zhang, C. Schmidt, J.A. Baller, N.V. Somia, A.J. Bogdanove, D.F. Voytas, Nucleic Acids Res. 39 (2011) e82. [21] E.L. Doyle, N.J. Booher, D.S. Standage, D.F. Voytas, V.P. Brendel, J.K. Vandyk, A.J. Bogdanove, Nucleic Acids Res. 40 (2012) W117–W122. [22] J. Valton, A. Dupuy, F. Daboussi, S. Thomas, A. Marechal, R. Macmaster, K. Melliand, A. Juillerat, P. Duchateau, J. Biol. Chem. 287 (2012) 38427–38432. [23] L. Cong, R. Zhou, Y.C. Kuo, M. Cunniff, F. Zhang, Nat. Commun. 3 (2012) 968. [24] J. Streubel, C. Blucher, A. Landgraf, J. Boch, Nat. Biotechnol. 30 (2012) 593–595. [25] D.A. Zacharias, J.D. Violin, A.C. Newton, R.Y. Tsien, Science 296 (2002) 913– 916. [26] A. Bolzer, G. Kreth, I. Solovei, D. Koehler, K. Saracoglu, C. Fauth, S. Muller, R. Eils, C. Cremer, M.R. Speicher, T. Cremer, PLoS Biol. 3 (2005) e157. [27] L.A. Boyer, T.I. Lee, M.F. Cole, S.E. Johnstone, S.S. Levine, J.P. Zucker, M.G. Guenther, R.M. Kumar, H.L. Murray, R.G. Jenner, D.K. Gifford, D.A. Melton, R. Jaenisch, R.A. Young, Cell 122 (2005) 947–956.
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