Programmable RNA recognition and cleavage by CRISPR/Cas9

O’Connell, Mitchell R.; Oakes, Benjamin L.; Sternberg, Samuel H.; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A.

doi:10.1038/nature13769

Cited by 573 publications

(484 citation statements)

References 35 publications

Supporting

Mentioning

465

Contrasting

Unclassified

Order By: Relevance

“…With the method described here, it also should be feasible to carry out dual-color labeling of two single-copy genomic loci using tiled arrays of sgRNAs across suitable regions of each locus and their different-colored dCas9 orthologs or, using the same strategy, between a single-copy locus and a repeated sequence lying nearby or more distant. In addition, Sp Cas9 was recently adapted for programmable RNA recognition and cleavage (22). The simultaneous use of Sp Cas9 for RNA recognition and other Cas9 orthologs for DNA recognition could provide a new tandem toolkit for studying the 4D nucleome and the regulation of eukaryotic gene expression across a broad landscape of cell types and stages of development, differentiation, and human disease.…”

Section: Discussionmentioning

confidence: 99%

Multicolor CRISPR labeling of chromosomal loci in human cells

Naseri

Reyes-Gutierrez

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

378

350

View full text Add to dashboard Cite

The intranuclear location of genomic loci and the dynamics of these loci are important parameters for understanding the spatial and temporal regulation of gene expression. Recently it has proven possible to visualize endogenous genomic loci in live cells by the use of transcription activator-like effectors (TALEs), as well as modified versions of the bacterial immunity clustered regularly interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Here we report the design of multicolor versions of CRISPR using catalytically inactive Cas9 endonuclease (dCas9) from three bacterial orthologs. Each pair of dCas9-fluorescent proteins and cognate single-guide RNAs (sgRNAs) efficiently labeled several target loci in live human cells. Using pairs of differently colored dCas9-sgRNAs, it was possible to determine the intranuclear distance between loci on different chromosomes. In addition, the fluorescence spatial resolution between two loci on the same chromosome could be determined and related to the linear distance between them on the chromosome's physical map, thereby permitting assessment of the DNA compaction of such regions in a live cell.4D nucleome | telomeres | pericentromeric DNA | chromosomes

show abstract

Section: Discussionmentioning

confidence: 99%

Multicolor CRISPR labeling of chromosomal loci in human cells

Naseri

Reyes-Gutierrez

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

378

350

View full text Add to dashboard Cite

show abstract

“…These results suggested that CASFISH could be a rapid and robust method for visualizing specific genomic elements in cells and primary tissue. We expect that a similar procedure could be developed for detection of RNA (15).…”

Section: Significancementioning

confidence: 99%

CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells

Deng

Shi

Tjian

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

250

214

View full text Add to dashboard Cite

Direct visualization of genomic loci in the 3D nucleus is important for understanding the spatial organization of the genome and its association with gene expression. Various DNA FISH methods have been developed in the past decades, all involving denaturing dsDNA and hybridizing fluorescent nucleic acid probes. Here we report a novel approach that uses in vitro constituted nuclease-deficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated caspase 9 (Cas9) complexes as probes to label sequence-specific genomic loci fluorescently without global DNA denaturation (Cas9-mediated fluorescence in situ hybridization, CASFISH). Using fluorescently labeled nuclease-deficient Cas9 (dCas9) protein assembled with various single-guide RNA (sgRNA), we demonstrated rapid and robust labeling of repetitive DNA elements in pericentromere, centromere, G-rich telomere, and coding gene loci. Assembling dCas9 with an array of sgRNAs tiling arbitrary target loci, we were able to visualize nonrepetitive genomic sequences. The dCas9/sgRNA binary complex is stable and binds its target DNA with high affinity, allowing sequential or simultaneous probing of multiple targets. CASFISH assays using differently colored dCas9/sgRNA complexes allow multicolor labeling of target loci in cells. In addition, the CASFISH assay is remarkably rapid under optimal conditions and is applicable for detection in primary tissue sections. This rapid, robust, less disruptive, and cost-effective technology adds a valuable tool for basic research and genetic diagnosis.

show abstract

“…These approaches are also incompatible with the use of denaturing isolation conditions. The CRISPR system was recently also used to establish a specific RNA isolation protocol with RNA-guided, biotin-tagged Cas9 under physiological conditions (O'Connell et al 2014). As this method also relies on the structural integrity and function of the Cas9 protein, it is incompatible with the highly stringent, denaturing conditions required to isolate direct RNA-protein interactions at high purity.…”

Section: Introductionmentioning

confidence: 99%

Specific RNP capture with antisense LNA/DNA mixmers

Rogell

Fischer

Rettel

et al. 2017

RNA

View full text Add to dashboard Cite

RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe "specific ribonucleoprotein (RNP) capture," a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein-RNA interactions taking place at "zero distance." Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins.

show abstract

Programmable RNA recognition and cleavage by CRISPR/Cas9

Cited by 573 publications

References 35 publications

Multicolor CRISPR labeling of chromosomal loci in human cells

Multicolor CRISPR labeling of chromosomal loci in human cells

CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells

Specific RNP capture with antisense LNA/DNA mixmers

Contact Info

Product

Resources

About