title: Programmable RNA Tracking in Live Cells with CRISPR/Cas9.
abstract: RNA-programmed genome editing using CRISPR/Cas9 from Streptococcus pyogenes has enabled rapid and accessible alteration of specific genomic loci in many organisms. A flexible means to target RNA would allow alteration and imaging of endogenous RNA transcripts analogous to CRISPR/Cas-based genomic tools, but most RNA targeting methods rely on incorporation of exogenous tags. Here, we demonstrate that nuclease-inactive S. pyogenes CRISPR/Cas9 can bind RNA in a nucleic-acid-programmed manner and allow endogenous RNA tracking in living cells. We show that nuclear-localized RNA-targeting Cas9 (RCas9) is exported to the cytoplasm only in the presence of sgRNAs targeting mRNA and observe accumulation of ACTB, CCNA2, and TFRC mRNAs in RNA granules that correlate with fluorescence in situ hybridization. We also demonstrate time-resolved measurements of ACTB mRNA trafficking to stress granules. Our results establish RCas9 as a means to track RNA in living cells in a programmable manner without genetically encoded tags.
authors.affiliation: Department of Cellular and Molecular Medicine and Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92037, USA; Materials Science and Engineering Graduate Program, University of California, San Diego, La Jolla, CA 92093, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Nelles
authors.affiliation: Department of Cellular and Molecular Medicine and Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92037, USA.
authors.forename: Mark Y
authors.initials: MY
authors.lastname: Fang
authors.affiliation: Department of Molecular and Cell Biology and Center for RNA Systems Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
authors.forename: Mitchell R
authors.initials: MR
authors.lastname: O'Connell
authors.affiliation: Department of Cellular and Molecular Medicine and Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92037, USA.
authors.forename: Jia L
authors.initials: JL
authors.lastname: Xu
authors.affiliation: Department of Cellular and Molecular Medicine and Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92037, USA.
authors.forename: Sebastian J
authors.initials: SJ
authors.lastname: Markmiller
authors.affiliation: Department of Molecular and Cell Biology and Center for RNA Systems Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Chemistry, Innovative Genomics Initiative and Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, Berkeley, CA 94720, USA.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Doudna
authors.affiliation: Department of Cellular and Molecular Medicine and Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92037, USA; Materials Science and Engineering Graduate Program, University of California, San Diego, La Jolla, CA 92093, USA; Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 1190777, Singapore. Electronic address: geneyeo@ucsd.edu.
authors.forename: Gene W
authors.initials: GW
authors.lastname: Yeo
mesh_terms: D064113:CRISPR-Cas Systems; D003594:Cytoplasmic Granules / Q000737:chemistry; D004720:Endonucleases / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000032:analysis; D006801:Humans; D012313:RNA / Q000032:analysis*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D012313:RNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D004720:Endonucleases