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      Comparison and optimization of CRISPR/dCas9/gRNA genome-labeling systems for live cell imaging

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          Abstract

          CRISPR/dCas9 binds precisely to defined genomic sequences through targeting of guide RNA (gRNA) sequences. In vivo imaging of genomic loci can be achieved by recruiting fluorescent proteins using either dCas9 or gRNA. We thoroughly validate and compare the effectiveness and specificity of several dCas9/gRNA genome labeling systems. Surprisingly, we discover that in the gRNA-labeling strategies, accumulation of tagged gRNA transcripts leads to non-specific labeling foci. Furthermore, we develop novel bimolecular fluorescence complementation (BIFC) methods that combine the advantages of both dCas9-labeling and gRNA-labeling strategies. The BIFC-dCas9/gRNA methods demonstrate high signal-to-noise ratios and have no non-specific foci.

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          CRISPR/Cas9 in Genome Editing and Beyond

          Haifeng Wang, Marie La Russa, Lei S Qi (2016)
          The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is emerging as a powerful tool for engineering the genome in diverse organisms. As an RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, and its development as a tool has made sequence-specific gene editing several magnitudes easier. The nuclease-deactivated form of Cas9 further provides a versatile RNA-guided DNA-targeting platform for regulating and imaging the genome, as well as for rewriting the epigenetic status, all in a sequence-specific manner. With all of these advances, we have just begun to explore the possible applications of Cas9 in biomedical research and therapeutics. In this review, we describe the current models of Cas9 function and the structural and biochemical studies that support it. We focus on the applications of Cas9 for genome editing, regulation, and imaging, discuss other possible applications and some technical considerations, and highlight the many advantages that CRISPR/Cas9 technology offers.
          Article 0 comments Cited 431 times – based on 0 reviews     Review now
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            Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow.

            Hanhui Ma, Li-Chun Tu, Ardalan Naseri … (2016)
            A lack of techniques to image multiple genomic loci in living cells has limited our ability to investigate chromosome dynamics. Here we describe CRISPRainbow, a system for labeling DNA in living cells based on nuclease-dead (d) Cas9 combined with engineered single guide RNA (sgRNA) scaffolds that bind sets of fluorescent proteins. We demonstrate simultaneous imaging of up to six chromosomal loci in individual live cells and document large differences in the dynamic properties of different chromosomal loci.
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              Versatile protein tagging in cells with split fluorescent protein

              Daichi Kamiyama, Sayaka Sekine, Benjamin Barsi-Rhyne … (2016)
              In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications.
              Article 0 comments Cited 179 times – based on 0 reviews     Review now
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                Author and article information

                Contributors
                Yu Zhang:
                ORCID: http://orcid.org/0000-0002-4888-5923
                zhangyu@nibs.ac.cn
                Journal
                Journal ID (nlm-ta): Genome Biol
                Journal ID (iso-abbrev): Genome Biol
                Title: Genome Biology
                Publisher: BioMed Central (London )
                ISSN (Print): 1474-7596
                ISSN (Electronic): 1474-760X
                Publication date (Electronic): 22 March 2018
                Publication date PMC-release: 22 March 2018
                Publication date Collection: 2018
                Volume: 19
                Electronic Location Identifier: 39
                Affiliations
                [1 ]ISNI 0000 0001 2256 9319, GRID grid.11135.37, Peking University-Tsinghua University-National Institute of Biological Sciences Joint Graduate Program, School of Life Sciences, , Peking University, ; Beijing, 100871 China
                [2 ]ISNI 0000 0001 0662 3178, GRID grid.12527.33, Graduate School of Peking Union Medical College, ; Beijing, 100730 China
                [3 ]ISNI 0000 0004 0644 5086, GRID grid.410717.4, National Institute of Biological Sciences, ; Beijing, 102206 China
                Author information
                http://orcid.org/0000-0002-4888-5923
                Article
                Publisher ID: 1413
                DOI: 10.1186/s13059-018-1413-5
                PMC ID: 5863892
                PubMed ID: 29566733
                SO-VID: 403d3850-3ce6-4049-8918-715a3c4c84c9
                Copyright © © The Author(s). 2018
                License:

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                Date received : 15 October 2017
                Date accepted : 28 February 2018
                Categories
                Subject: Method
                Custom metadata
                issue-copyright-statement © The Author(s) 2018

                ScienceOpen disciplines: Genetics
                Keywords: genome labeling,crispr/dcas9,bimolecular fluorescence complementation (bifc)
                Data availability:
                ScienceOpen disciplines: Genetics
                Keywords: genome labeling, crispr/dcas9, bimolecular fluorescence complementation (bifc)

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