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 inScience (American Association for the Advancement of Science) Vol. 359; no. 6379; pp. 1050 - 1055
Main Authors Gu, Bo, Swigut, Tomek, Spencley, Andrew, Bauer, Matthew R, Chung, Mingyu, Meyer, Tobias, Wysocka, Joanna
Format Journal Article
LanguageEnglish
Published United States The American Association for the Advancement of Science 02.03.2018
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.
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B.G., T.S., and J.W. conceived and designed the study; B.G. performed experiments with help from M.R.B.; A.S. conducted and analyzed the ChIP-qPCR assay; M.C. assisted with smFISH matlab script modification and implementation; T.M. and T.S. provided critical advice on experimental designs, data analyses, and data interpretations; J.W. supervised the project; and B.G. and J.W. wrote the manuscript with input from all coauthors.
Author contributions
ISSN:0036-8075
1095-9203
DOI:10.1126/science.aao3136