CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging

Ma, Hanhui; Tu, Li‐Chun; Naseri, Ardalan; Chung, Yu-Chieh; Grünwald, David; Zhang, Shaojie; Pederson, Thoru

doi:10.1038/s41592-018-0174-0

Cited by 136 publications

(182 citation statements)

References 31 publications

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“…The CRISPR/Cas systems offer versatile DNA binding proteins such as Cas9 that can be programmable by a guide RNA (gRNA) for a specific target and has revolutionized the field of genome engineering and genomics (11). Fluorescent proteins recruited by dCas9 or assembled on the gRNA scaffold offer easy-to-use platforms for live cell genomic imaging (12)(13)(14)(15)(16)(17). 20…”

Section: Main Textmentioning

confidence: 99%

CRISPR-mediated Multiplexed Live Cell Imaging of Nonrepetitive Genomic Loci

Clow

Jillette

Zhu

et al. 2020

Preprint

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Three-dimensional (3D) structures of the genome are dynamic, heterogeneous and functionally important. Live cell imaging has become the leading method for chromatin dynamics tracking. However, existing CRISPR-and TALE-based genomic labeling techniques have been hampered by laborious protocols and low signal-to-noise ratios (SNRs), and are thus mostly applicable to repetitive sequences. Here, we report a versatile CRISPR/Casilio-based 20 imaging method, with an enhanced SNR, that allows for one nonrepetitive genomic locus to be labeled using a single sgRNA. We constructed Casilio dual-color probes to visualize the dynamic interactions of cohesin-bound elements in single live cells. By forming a binary sequence of multiple Casilio probes (PISCES) across a continuous stretch of DNA, we track the dynamic 3D folding of a 74kb genomic region over time. This method offers unprecedented resolution and 25 scalability for delineating the dynamic 4D nucleome.One Sentence Summary: Casilio enables multiplexed live cell imaging of nonrepetitive DNA loci for illuminating the real-time dynamics of genome structures.

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Section: Main Textmentioning

confidence: 99%

CRISPR-mediated Multiplexed Live Cell Imaging of Nonrepetitive Genomic Loci

Clow

Jillette

Zhu

et al. 2020

Preprint

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“…Some potential applications of JACKIE pipeline are illustrated in Fig 2. For example, highly repetitive sequences (>30 copies) and low repetitive arrays (~6 copies) can be used for imaging applications (Fig 2a) (Cheng, et al, 2016; Ma, et al, 2018; Maass, et al, 2018; Qin, et al, 2017). These sites can be identified from JACKIE output by filtering for the desired range of repeat number and span size.…”

Section: Use Casesmentioning

confidence: 99%

“…CRISPR/Cas technologies have revolutionized genome research by providing versatile tools for “writing” the genome, epigenome and transcriptome as well as “reading” the dynamics of the genome through live cell genomic imaging techniques (Sander and Joung, 2014). Genomic imaging by CRISPR/Cas-based technologies require clustered repetitive sequences (Cheng, et al, 2016; Ma, et al, 2018; Maass, et al, 2018; Qin, et al, 2017). dCas9-based gene activation and epigenome editing also benefited from synergistic binding events (Cheng, et al, 2016; Cheng, et al, 2013; Maeder, et al, 2013; Mali, et al, 2013; Taghbalout, et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

JACKIE: Fast enumeration of genomic single- and multi-copy target sites and their off-targets for CRISPR and other engineered nuclease systems

Cheng

2020

Preprint

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11Summary: CRISPR-based methods for genome, epigenome editing and imaging have provided 12 powerful tools to interrogate the functions of the genome. The design of guide RNA (gRNA) is a 13 vital step of CRISPR experiments. We report here the implementation of JACKIE (Jackie and 14 Albert's CRISPR K-mer Instances Enumerator), a pipeline for enumerating all potential single-15 and multi-copy CRISPR sites in the genome. We demonstrate the application of JACKIE to 16 identify locus-specific repetitive sequences for CRISPR/Casilio-based genomic labeling. 17 18 Availability: Source codes and CRISPR site databases (JACKIEdb) for hg38 and mm10 are 19 available for download at http://crispr.software/JACKIE 20 21 118 ./, and score field recording the copy number.119 Parallelization is achieved by either starting separate job on individual <6merPrefix>.bin files, or 120 by starting batch jobs focusing on 2mer prefices (e.g., AA AT AC AG operating on AA*.bin, 121 AT*.bin, AC*.bin, AG*.bin, etc). The outputs (<6merPrefix>.bed) are then merged into a 122 combined bed file using cat unix command. Helper scripts are provided for downstream 123 processing of the bed file into collapsed bed file with each record encoding all binding sites of 124 6 the same sequence in the same chromosome, for extracting binding sites within defined 125 regions, or for running the Cas-OFFinder program to identify off-target profiles of selected 126 sequences. 127 Fig 2. Potential applications of JACKIE pipeline. (a) JACKIE can be used to identify 128 clustered CRISPR binding sites with high copy number or low copy number for CRISPR-based 129 genomic imaging experiments. (b) To demonstate genomic imaging use case, we filtered 130 sgRNA sequences with >30 copies clustered within specific <10kb regions and performed 131 Casilio imaging experiment using designed sgRNAs. UCSC genome browser screenshots are 132 shown on the left with the line-bar annotating the CRISPR sites, and fluorescence microscopy 133 images shown on the right. Microscopy images were derived by merging green (Casilio-134 labeling) and blue (DAPI: nucleus stain). Arrows point to fluorescent loci of interest. Scale bars 135 indicate 5μm. (c) JACKIE can be used to identify clustered CRISPR binding sites for synergistic 136 activation or repression of target cis-regulatory elements (e.g., promoters) using CRISPR-based 137 epigenetic editing approaches (e.g., CRISPRa or CRISPRi). (d) JACKIE can identify unique 138 CRISPR binding sites in the genome for precise indel induction, dual CRISPR sites for targeted 139 deletion, or clustered sites spanning a genomic region to induce complex rearrangement 140 events.

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“…OligoSTORM, MERFISH, Hi-M or ORCA has been applied to trace DNA folding [12][13][14][15][16][17][18] in fixed cells. On the other hand, CRISPR-based imaging provides a versatile and powerful tool to track chromatin topology in live cells in real time [19,20] .…”

Section: Introductionmentioning

confidence: 99%

Simultaneous Epigenetic Perturbation and Genome Imaging Reveal Distinct Roles of H3K9me3 in Chromatin Architecture and Transcription

Feng

Wang

et al. 2020

Preprint

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Despite the long-observed correlation between H3K9me3, chromatin architecture and transcriptional repression, how H3K9me3 regulates genome higher-order organization and transcriptional activity in living cells remains unclear. Here we develop EpiGo (Epigenetic perturbation induced Genome organization)-KRAB to introduce H3K9me3 at hundreds of loci spanning megabases on human chromosome 19 and simultaneously track genome organization. EpiGo-KRAB is sufficient to induce de novo heterochromatin-like domain formation, which requires SETDB1, a methyltransferase of H3K9me3. Unexpectedly, EpiGo-KRAB induced heterochromatin-like domain does not result in widespread gene repression except a small set of genes with concurrent loss of H3K4me3 and H3K27ac. Ectopic H3K9me3 appears to spread in inactive regions but is largely restricted to transcriptional initiation sites in active regions. Finally, Hi-C analysis showed that EpiGo-KRAB induced to reshape existing compartments. These results reveal the role of H3K9me3 in genome organization could be partially separated from its function in gene repression.

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CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging

Cited by 136 publications

References 31 publications

CRISPR-mediated Multiplexed Live Cell Imaging of Nonrepetitive Genomic Loci

CRISPR-mediated Multiplexed Live Cell Imaging of Nonrepetitive Genomic Loci

JACKIE: Fast enumeration of genomic single- and multi-copy target sites and their off-targets for CRISPR and other engineered nuclease systems

Simultaneous Epigenetic Perturbation and Genome Imaging Reveal Distinct Roles of H3K9me3 in Chromatin Architecture and Transcription

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