title: Live cell imaging of low- and non-repetitive chromosome loci using CRISPR-Cas9.
abstract: Imaging chromatin dynamics is crucial to understand genome organization and its role in transcriptional regulation. Recently, the RNA-guidable feature of CRISPR-Cas9 has been utilized for imaging of chromatin within live cells. However, these methods are mostly applicable to highly repetitive regions, whereas imaging regions with low or no repeats remains as a challenge. To address this challenge, we design single-guide RNAs (sgRNAs) integrated with up to 16 MS2 binding motifs to enable robust fluorescent signal amplification. These engineered sgRNAs enable multicolour labelling of low-repeat-containing regions using a single sgRNA and of non-repetitive regions with as few as four unique sgRNAs. We achieve tracking of native chromatin loci throughout the cell cycle and determine differential positioning of transcriptionally active and inactive regions in the nucleus. These results demonstrate the feasibility of our approach to monitor the position and dynamics of both repetitive and non-repetitive genomic regions in live cells.
authors.affiliation: Department of Physics, University of California at Berkeley, Berkeley, California 94720, USA.
authors.forename: Peiwu
authors.initials: P
authors.lastname: Qin
authors.affiliation: Department of Biochemistry and Molecular Genetics, School of Medicine, University of Virginia, Charlottesville, Virginia 22908, USA.
authors.forename: Mahmut
authors.initials: M
authors.lastname: Parlak
authors.affiliation: Department of Biochemistry and Molecular Genetics, School of Medicine, University of Virginia, Charlottesville, Virginia 22908, USA.
authors.forename: Cem
authors.initials: C
authors.lastname: Kuscu
authors.affiliation: Department of Physics, University of California at Berkeley, Berkeley, California 94720, USA.
authors.forename: Jigar
authors.initials: J
authors.lastname: Bandaria
authors.affiliation: Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA.
authors.forename: Mustafa
authors.initials: M
authors.lastname: Mir
authors.affiliation: Department of Biochemistry and Molecular Genetics, School of Medicine, University of Virginia, Charlottesville, Virginia 22908, USA.
authors.forename: Karol
authors.initials: K
authors.lastname: Szlachta
authors.affiliation: Department of Biochemistry and Molecular Genetics, School of Medicine, University of Virginia, Charlottesville, Virginia 22908, USA.|Department of Computer Science, University of Virginia, Charlottesville, Virginia 22904, USA.
authors.forename: Ritambhara
authors.initials: R
authors.lastname: Singh
authors.affiliation: Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA.
authors.forename: Xavier
authors.initials: X
authors.lastname: Darzacq
authors.affiliation: Department of Physics, University of California at Berkeley, Berkeley, California 94720, USA.|Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA.
authors.forename: Ahmet
authors.initials: A
authors.lastname: Yildiz
authors.affiliation: Department of Biochemistry and Molecular Genetics, School of Medicine, University of Virginia, Charlottesville, Virginia 22908, USA.
authors.forename: Mazhar
authors.initials: M
authors.lastname: Adli
mesh_terms: D064113:CRISPR-Cas Systems; D002453:Cell Cycle / Q000235:genetics*; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D002843:Chromatin / Q000378:metabolism* / Q000648:ultrastructure; D005240:Feasibility Studies; D056426:Genetic Loci; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D000069416:Intravital Microscopy; D018613:Microscopy, Confocal; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000166:cytology
chemical_list: D002843:Chromatin; D000094704:RNA, Guide, CRISPR-Cas Systems