Abstract
The biological function of chromatin bases on its spatial organization and dynamic activities in different situations. Labeling and tracing of genomic sequences has been a huge challenge in studying the spatial dynamics of chromatin. We reported the development of ‘all-in-one Casilio system (Aio-Casilio)’, a new system that enables the labeling of endogenous genomic loci. The Aio-Casilio system consists of the dCas9 protein, mClover fused with the Pumilio and FBF proteins RNA-binding domain (PUF domain) and an U6-sgRNA appended with multiple PUF-binding site(s). Here we showed that the Aio-Casilio system is robust tool for imaging of repetitive elements in telomeres and major satellite in N2A cells. Furthermore, we developed a PBTon–Aio-Casilio System, which enables the visualization of repetitive sequences in mES cells. However, this system failed to establish a labeled ES cell line when we attempted to establish a stable labeling cell line for living cell image. This Aio-Casilio imaging tool has potential to significantly improve the capacity to study the conformation of chromosomes in living cells.
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Abil Z, Denard CA, Zhao H (2014) Modular assembly of designer PUF proteins for specific post-transcriptional regulation of endogenous RNA. J Biol Eng 8:7. doi:10.1186/1754-1611-8-7
Anton T, Bultmann S, Leonhardt H, Markaki Y (2014) Visualization of specific DNA sequences in living mouse embryonic stem cells with a programmable fluorescent CRISPR/Cas system. Nucleus 5:163–172. doi:10.4161/nucl.28488
Barrangou R et al (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709–1712. doi:10.1126/science.1138140
Belmont AS, Li G, Sudlow G, Robinett C (1999) Visualization of large-scale chromatin structure and dynamics using the lac operator/lac repressor reporter system. Methods Cell Biol 58:203–222
Chen Y, Varani G (2013) Engineering RNA-binding proteins for biology. FEBS J 280:3734–3754. doi:10.1111/febs.12375
Chen B et al (2013) Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155:1479–1491. doi:10.1016/j.cell.2013.12.001
Cheng AW et al (2013) Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res 23:1163–1171 doi:10.1038/cr.2013.122
Cheng AW et al (2016) Casilio: a versatile CRISPR–Cas9–Pumilio hybrid for gene regulation and genomic labeling. Cell Res 26:254–257. doi:10.1038/cr.2016.3
Christian M et al (2010) Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186:757–761. doi:10.1534/genetics.110.120717
Cong L et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823. doi:10.1126/science.1231143
Cui C, Shu W, Li P (2016) Fluorescence in situ hybridization: cell-based genetic diagnostic and research applications. Front Cell Dev Biol 4:89 doi:10.3389/fcell.2016.00089
Deng W, Shi X, Tjian R, Lionnet T, Singer RH (2015) CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells. Proc Natl Acad Sci USA 112:11870–11875. doi:10.1073/pnas.1515692112
Gilbert LA et al (2013) CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154:442–451. doi:10.1016/j.cell.2013.06.044
Jiang W, Marraffini LA (2015) CRISPR-Cas: new tools for genetic manipulations from bacterial immunity systems. Annu Rev Microbiol 69:209–228. doi:10.1146/annurev-micro-091014-104441
Konermann S et al (2013) Optical control of mammalian endogenous transcription and epigenetic states. Nature 500:472–476. doi:10.1038/nature12466
Lindhout BI, Fransz P, Tessadori F, Meckel T, Hooykaas PJ, van der Zaal BJ (2007) Live cell imaging of repetitive DNA sequences via GFP-tagged polydactyl zinc finger proteins. Nucleic Acids Res 35:e107. doi:10.1093/nar/gkm618
Ma H, Naseri A, Reyes-Gutierrez P, Wolfe SA, Zhang S, Pederson T (2015) Multicolor CRISPR labeling of chromosomal loci in human cells. Proc Natl Acad Sci USA 112:3002–3007. doi:10.1073/pnas.1420024112
Miller JC et al (2007) An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25:778–785. doi:10.1038/nbt1319
Miyanari Y, Ziegler-Birling C, Torres-Padilla ME (2013) Live visualization of chromatin dynamics with fluorescent TALEs. Nat Struct Mol Biol 20:1321–1324 doi:10.1038/nsmb.2680
Park TS, Han JY (2012) piggyBac transposition into primordial germ cells is an efficient tool for transgenesis in chickens. Proc Natl Acad Sci USA 109:9337–9341. doi:10.1073/pnas.1203823109
Perez EE et al (2008) Establishment of HIV-1 resistance in CD4+T cells by genome editing using zinc-finger nucleases. Nat Biotechnol 26:808–816. doi:10.1038/nbt1410
Perez-Pinera P et al (2013) RNA-guided gene activation by CRISPR–Cas9-based transcription factors. Nat Methods 10:973–976. doi:10.1038/nmeth.2600
Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152:1173–1183. doi:10.1016/j.cell.2013.02.022
Qin P et al (2017) Live cell imaging of low- and non-repetitive chromosome loci using CRISPR–Cas9. Nat Commun 8:14725 doi:10.1038/ncomms14725
Wang S, Su JH, Zhang F, Zhuang X (2016) An RNA-aptamer-based two-color CRISPR labeling system. Sci Rep 6:26857 doi:10.1038/srep26857
Yuan M, Webb E, Lemoine NR, Wang Y (2016) CRISPR–Cas9 as a powerful tool for efficient creation of oncolytic viruses. Viruses 8:72. doi:10.3390/v8030072
Zhang F, Cong L, Lodato S, Kosuri S, Church GM, Arlotta P (2011) Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat Biotechnol 29:149–153. doi:10.1038/nbt.1775
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Zhang, S., Song, Z. Aio-Casilio: a robust CRISPR–Cas9–Pumilio system for chromosome labeling. J Mol Hist 48, 293–299 (2017). https://doi.org/10.1007/s10735-017-9727-2
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DOI: https://doi.org/10.1007/s10735-017-9727-2