CRISPR-Cas9 nuclear dynamics and target recognition in living cells

Ma, Hanhui; Tu, Li‐Chun; Naseri, Ardalan; Huisman, Maximiliaan; Zhang, Shaojie; Grünwald, David; Pederson, Thoru

doi:10.1083/jcb.201604115

Cited by 190 publications

(194 citation statements)

References 38 publications

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“…The Shield1- and doxycycline-inducible dSpyCas9-mCherry-APEX2 construct was made by subcloning Flag-APEX2 from Flag-APEX2-NES (Addgene 49386) into DD-dSpyCas9-mCherry 21 using the pHAGE backbone. Two additional NLSs (SV40 and nucleoplasmin NLS) were inserted at each terminus to improve nuclear localization.…”

Section: Methodsmentioning

confidence: 99%

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C-BERST: defining subnuclear proteomic landscapes at genomic elements with dCas9–APEX2

et al. 2018

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Mapping proteomic composition at distinct genomic loci in living cells has been a long-standing challenge. Here we report that dCas9-APEX2 Biotinylation at genomic Elements by Restricted Spatial Tagging (C-BERST) allows the rapid, unbiased mapping of proteomes near defined genomic loci, as demonstrated for telomeres and centromeres. C-BERST enables the high-throughput identification of proteins associated with specific sequences, facilitating annotation of these factors and their roles in nuclear biology.

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Section: Methodsmentioning

confidence: 99%

“…Lentiviral transduction was as described 21 . Six-fold higher titers of sgRNA-encoding lentiviruses were used for transduction relative to dSpyCas9-APEX2 lentivirus.…”

Section: Methodsmentioning

confidence: 99%

C-BERST: defining subnuclear proteomic landscapes at genomic elements with dCas9–APEX2

et al. 2018

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“…143 More recently, they have been adapted to allow high-resolution super-resolution imaging of up to 30 genomic loci using short oligonucleotide probes 144 or up to 6 loci using modified CRISPR-based systems targeted to the genome by engineered guide RNAs. 145,146 Although both approaches have their drawbacks, they represent significant improvements over traditional FISH analysis yet can still detect only a tiny fraction of the genomewide contacts seen with Hi-C. 144,[147][148][149][150] However, improvements and innovative new approaches are certainly on the horizon. Optical imaging can also capture chromosome dynamics and 3D positioning of loci in live cells, neither of which can be determined using static…”

Section: Future Perspectivesmentioning

confidence: 99%

The biology and polymer physics underlying large‐scale chromosome organization

Sazer

Schiessel

2017

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Chromosome large‐scale organization is a beautiful example of the interplay between physics and biology. DNA molecules are polymers and thus belong to the class of molecules for which physicists have developed models and formulated testable hypotheses to understand their arrangement and dynamic properties in solution, based on the principles of polymer physics. Biologists documented and discovered the biochemical basis for the structure, function and dynamic spatial organization of chromosomes in cells. The underlying principles of chromosome organization have recently been revealed in unprecedented detail using high‐resolution chromosome capture technology that can simultaneously detect chromosome contact sites throughout the genome. These independent lines of investigation have now converged on a model in which DNA loops, generated by the loop extrusion mechanism, are the basic organizational and functional units of the chromosome.

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“…Single-molecule experiments have demonstrated that Cas9 initiates the target DNA search process by probing for a proper PAM sequence before interrogating the flanking DNA for potential guide RNA complementarity (94). Target recognition occurs through three-dimensional collisions, in which Cas9 rapidly dissociates from DNA that does not contain the appropriate PAM sequence, and dwell time depends on the complementarity between guide RNA and adjacent DNA when a proper PAM is present (55,61,94). Once Cas9 has found a target site with the appropriate PAM, it triggers local DNA melting at the PAM-adjacent nucleation site, followed by RNA strand invasion to form an RNA-DNA hybrid and a displaced DNA strand (termed R-loop) from PAM-proximal to PAM-distal ends (94,96).…”

Section: Target Search and Recognitionmentioning

confidence: 99%

CRISPR–Cas9 Structures and Mechanisms

Jiang

Doudna

2017

Annu. Rev. Biophys.

1,560

1,206

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Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes. The use of CRISPR-Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.

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CRISPR-Cas9 nuclear dynamics and target recognition in living cells

Cited by 190 publications

References 38 publications

C-BERST: defining subnuclear proteomic landscapes at genomic elements with dCas9–APEX2

C-BERST: defining subnuclear proteomic landscapes at genomic elements with dCas9–APEX2

The biology and polymer physics underlying large‐scale chromosome organization

CRISPR–Cas9 Structures and Mechanisms

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