Transcription-coupled changes in nuclear mobility of mammalian cis-regulatory elements

To achieve guide RNA (gRNA) multiplexing and an efficient delivery of tens of distinct gRNAs into single cells, we developed a molecular assembly strategy termed chimeric array of gRNA oligonucleotides (CARGO). We coupled CARGO with dCas9 (catalytically dead Cas9) imaging to quantitatively measure t...

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Bibliographic Details
Published in:Science (American Association for the Advancement of Science) 2018-03, Vol.359 (6379), p.1050-1055
Main Authors: Gu, Bo, Swigut, Tomek, Spencley, Andrew, Bauer, Matthew R, Chung, Mingyu, Meyer, Tobias, Wysocka, Joanna
Format: Article
Language:English
Subjects:
Animals
Bacterial Proteins
Cargo
Cell differentiation
Cell Line
Cell Nucleus - genetics
Cells (biology)
CRISPR-Associated Protein 9
Deoxyribonucleic acid
Differentiation (biology)
DNA
DNA-directed RNA polymerase
Embryo cells
Endonucleases
Enhancers
Gene regulation
gRNA
Individualized Instruction
Loci
Mice
Mobility
Multiplexing
Nuclei
Nuclei (cytology)
Oligonucleotide Array Sequence Analysis
Oligonucleotides
Promoters
Regulatory sequences
Regulatory Sequences, Nucleic Acid
Ribonucleic acid
RNA
RNA polymerase II
RNA Polymerase II - metabolism
RNA, Guide, CRISPR-Cas Systems
Single Molecule Imaging - methods
Single-Cell Analysis - methods
Stem cells
Transcription activation
Transcription, Genetic
Transcriptional Activation
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Summary:To achieve guide RNA (gRNA) multiplexing and an efficient delivery of tens of distinct gRNAs into single cells, we developed a molecular assembly strategy termed chimeric array of gRNA oligonucleotides (CARGO). We coupled CARGO with dCas9 (catalytically dead Cas9) imaging to quantitatively measure the movement of enhancers and promoters that undergo differentiation-associated activity changes in live embryonic stem cells. Whereas all examined functional elements exhibited subdiffusive behavior, their relative mobility increased concurrently with transcriptional activation. Furthermore, acute perturbation of RNA polymerase II activity can reverse these activity-linked increases in loci mobility. Through quantitative CARGO-dCas9 imaging, we provide direct measurements of cis-regulatory element dynamics in living cells and distinct cellular and activity states and uncover an intrinsic connection between cis-regulatory element mobility and transcription.
ISSN:0036-8075
1095-9203
DOI:10.1126/science.aao3136