title: An RNA-aptamer-based two-color CRISPR labeling system.
abstract: The spatial organization and dynamics of chromatin play important roles in essential biological functions. However, direct visualization of endogenous genomic loci in living cells has proven to be laborious until the recent development of CRISPR-Cas9-based chromatin labeling methods. These methods rely on the recognition of specific DNA sequences by CRISPR single-guide RNAs (sgRNAs) and fluorescent-protein-fused catalytically inactive Cas9 to label specific chromatin loci in cells. Previously, multicolor chromatin labeling has been achieved using orthogonal Cas9 proteins from different bacterial species fused to different fluorescent proteins. Here we report the development of an alternative two-color CRISPR labeling method using only the well-characterized Streptococcus pyogenes Cas9, by incorporating MS2 or PP7 RNA aptamers into the sgRNA. The MS2 or PP7 aptamers then recruit the corresponding MS2 or PP7 coat proteins fused with different fluorescent proteins to the target genomic loci. Here we demonstrate specific and orthogonal two-color labeling of repetitive sequences in living human cells using this method. By attaching the MS2 or PP7 aptamers to different locations on the sgRNA, we found that extending the tetraloop and stem loop 2 of the sgRNA with MS2 or PP7 aptamers enhances the signal-to-background ratio of chromatin imaging.
authors.affiliation: Howard Hughes Medical Institute, Cambridge, MA 02138, USA.|Department of Chemistry and Chemical Biology and Department of Physics, Harvard University, Cambridge, MA 02138, USA.
authors.forename: Siyuan
authors.initials: S
authors.lastname: Wang
authors.affiliation: Howard Hughes Medical Institute, Cambridge, MA 02138, USA.|Department of Chemistry and Chemical Biology and Department of Physics, Harvard University, Cambridge, MA 02138, USA.
authors.forename: Jun-Han
authors.initials: JH
authors.lastname: Su
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.|McGovern Institute for Brain Research, MIT, Cambridge, MA 02139, USA.|Department of Brain and Cognitive Sciences and Department of Biological Engineering, MIT, Cambridge, MA 02139, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Howard Hughes Medical Institute, Cambridge, MA 02138, USA.|Department of Chemistry and Chemical Biology and Department of Physics, Harvard University, Cambridge, MA 02138, USA.
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Zhuang
mesh_terms: D052157:Aptamers, Nucleotide / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D045744:Cell Line, Tumor; D002843:Chromatin / Q000378:metabolism*; D004720:Endonucleases / Q000235:genetics; D005455:Fluorescent Antibody Technique / Q000379:methods; D056426:Genetic Loci; D006801:Humans; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D052157:Aptamers, Nucleotide; D001426:Bacterial Proteins; D002843:Chromatin; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases