Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Nucleosome breathing and remodeling constrain CRISPR-Cas9 function.

ABSTRACT: The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo.

SUBMITTER: Isaac RS

PROVIDER: S-EPMC4880442 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Nucleosome breathing and remodeling constrain CRISPR-Cas9 function.

Isaac R Stefan RS Jiang Fuguo F Doudna Jennifer A JA Lim Wendell A WA Narlikar Geeta J GJ Almeida Ricardo R

eLife 20160428

The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas ...[more]

PMID: 27130520

Similar Datasets

The impact of nucleosome structure on CRISPR/Cas9 fidelity.

Project description:The clustered regularly interspaced short palindromic repeats (CRISPR) Cas system is a powerful tool that has the potential to become a therapeutic gene editor in the near future. Cas9 is the best studied CRISPR system and has been shown to have problems that restrict its use in therapeutic applications. Chromatin structure is a known impactor of Cas9 targeting and there is a gap in knowledge on Cas9's efficacy when targeting such locations. To quantify at a single base pair resolution how chromatin inhibits on-target gene editing relative to off-target editing of exposed mismatching targets, we developed the gene editor mismatch nucleosome inhibition assay (GEMiNI-seq). GEMiNI-seq utilizes a library of nucleosome sequences to examine all target locations throughout nucleosomes in a single assay. The results from GEMiNI-seq revealed that the location of the protospacer-adjacent motif (PAM) sequence on the nucleosome edge drives the ability for Cas9 to access its target sequence. In addition, Cas9 had a higher affinity for exposed mismatched targets than on-target sequences within a nucleosome. Overall, our results show how chromatin structure impacts the fidelity of Cas9 to potential targets and highlight how targeting sequences with exposed PAMs could limit off-target gene editing, with such considerations improving Cas9 efficacy and resolving current limitations.

| S-EPMC10018339 | biostudies-literature

Single-nucleosome imaging unveils that condensins and nucleosome-nucleosome interactions differentially constrain chromatin to organize mitotic chromosomes.

Project description:For accurate mitotic cell division, replicated chromatin must be assembled into chromosomes and faithfully segregated into daughter cells. While protein factors like condensin play key roles in this process, it is unclear how chromosome assembly proceeds as molecular events of nucleosomes in living cells and how condensins act on nucleosomes to organize chromosomes. To approach these questions, we investigate nucleosome behavior during mitosis of living human cells using single-nucleosome tracking, combined with rapid-protein depletion technology and computational modeling. Our results show that local nucleosome motion becomes increasingly constrained during mitotic chromosome assembly, which is functionally distinct from condensed apoptotic chromatin. Condensins act as molecular crosslinkers, locally constraining nucleosomes to organize chromosomes. Additionally, nucleosome-nucleosome interactions via histone tails constrain and compact whole chromosomes. Our findings elucidate the physical nature of the chromosome assembly process during mitosis.

| S-EPMC11339268 | biostudies-literature

The nucleosome remodeling factor.

Project description:An essential component of the chromatin remodeling machinery is NURF (Nucleosome Remodeling Factor), the founding member of the ISWI family of chromatin remodeling complexes. In vertebrates and invertebrates alike, NURF has many important functions in chromatin biology including regulating transcription, establishing boundary elements, and promoting higher order chromatin structure. Since NURF is essential to many aspects of chromatin biology, knowledge of its function is required to fully understand how the genome is regulated. This review will summarize what is currently known of its biological functions, conservation in the most prominent model organisms, biochemical functions as a nucleosome remodeling enzyme, and its possible relevance to human cancer.

| S-EPMC4839296 | biostudies-literature

A Genome-wide CRISPR Screen Reveals a Role for the Non-canonical Nucleosome-Remodeling BAF Complex in Foxp3 Expression and Regulatory T Cell Function.

Project description:Regulatory T (Treg) cells play a pivotal role in suppressing auto-reactive T cells and maintaining immune homeostasis. Treg cell development and function are dependent on the transcription factor Foxp3. Here, we performed a genome-wide CRISPR loss-of-function screen to identify Foxp3 regulators in mouse primary Treg cells. Foxp3 regulators were enriched in genes encoding subunits of the SWI/SNF nucleosome-remodeling and SAGA chromatin-modifying complexes. Among the three SWI/SNF-related complexes, the Brd9-containing non-canonical (nc) BAF complex promoted Foxp3 expression, whereas the PBAF complex was repressive. Chemical-induced degradation of Brd9 led to reduced Foxp3 expression and reduced Treg cell function in vitro. Brd9 ablation compromised Treg cell function in inflammatory disease and tumor immunity in vivo. Furthermore, Brd9 promoted Foxp3 binding and expression of a subset of Foxp3 target genes. Our findings provide an unbiased analysis of the genetic networks regulating Foxp3 and reveal ncBAF as a target for therapeutic manipulation of Treg cell function.

| S-EPMC7341821 | biostudies-literature

Replication-guided nucleosome packing and nucleosome breathing expedite the formation of dense arrays.

Project description:The first level of genome packaging in eukaryotic cells involves the formation of dense nucleosome arrays, with DNA coverage near 90% in yeasts. How cells achieve such high coverage within a short time, e.g. after DNA replication, remains poorly understood. It is known that random sequential adsorption of impenetrable particles on a line reaches high density extremely slowly, due to a jamming phenomenon. The nucleosome-shifting action of remodeling enzymes has been proposed as a mechanism to resolve such jams. Here, we suggest two biophysical mechanisms which assist rapid filling of DNA with nucleosomes, and we quantitatively characterize these mechanisms within mathematical models. First, we show that the 'softness' of nucleosomes, due to nucleosome breathing and stepwise nucleosome assembly, significantly alters the filling behavior, speeding up the process relative to 'hard' particles with fixed, mutually exclusive DNA footprints. Second, we explore model scenarios in which the progression of the replication fork could eliminate nucleosome jamming, either by rapid filling in its wake or via memory of the parental nucleosome positions. Taken together, our results suggest that biophysical effects promote rapid nucleosome filling, making the reassembly of densely packed nucleosomes after DNA replication a simpler task for cells than was previously thought.

| S-EPMC4267636 | biostudies-literature

Mouse genome engineering via CRISPR-Cas9 for study of immune function.

Project description:Clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas9) technology has proven a formidable addition to our armory of approaches for genomic editing. Derived from pathways in archaea and bacteria that mediate the resistance to exogenous genomic material, the CRISPR-Cas9 system utilizes a short single guide RNA (sgRNA) to direct the endonuclease Cas9 to virtually anywhere in the genome. Upon targeting, Cas9 generates DNA double-strand breaks (DSBs) and facilitates the repair or insertion of mutations, insertion of recombinase recognition sites, or large DNA elements. Here, we discuss the practical advantages of the CRISPR-Cas9 system over conventional and other nuclease-based targeting technologies and provide suggestions for the use of this technology to address immunological questions.

| S-EPMC4720985 | biostudies-literature

Nucleosome remodeling by hMSH2-hMSH6.

Project description:DNA nucleotide mismatches and lesions arise on chromosomes that are a complex assortment of protein and DNA (chromatin). The fundamental unit of chromatin is a nucleosome that contains approximately 146 bp DNA wrapped around an H2A, H2B, H3, and H4 histone octamer. We demonstrate that the mismatch recognition heterodimer hMSH2-hMSH6 disassembles a nucleosome. Disassembly requires a mismatch that provokes the formation of hMSH2-hMSH6 hydrolysis-independent sliding clamps, which translocate along the DNA to the nucleosome. The rate of disassembly is enhanced by actual or mimicked acetylation of histone H3 within the nucleosome entry-exit and dyad axis that occurs during replication and repair in vivo and reduces DNA-octamer affinity in vitro. Our results support a passive mechanism for chromatin remodeling whereby hMSH2-hMSH6 sliding clamps trap localized fluctuations in nucleosome positioning and/or wrapping that ultimately leads to disassembly, and highlight unanticipated strengths of the Molecular Switch Model for mismatch repair (MMR).

| S-EPMC3010363 | biostudies-literature

Metagenomic reconstructions of bacterial CRISPR loci constrain population histories.

Project description:Bacterial CRISPR-Cas systems provide insight into recent population history because they rapidly incorporate, in a unidirectional manner, short fragments (spacers) from coexisting infective virus populations into host chromosomes. Immunity is achieved by sequence identity between transcripts of spacers and their targets. Here, we used metagenomics to study the stability and dynamics of the type I-E CRISPR-Cas locus of Leptospirillum group II bacteria in biofilms sampled over 5 years from an acid mine drainage (AMD) system. Despite recovery of 452,686 spacers from CRISPR amplicons and metagenomic data, rarefaction curves of spacers show no saturation. The vast repertoire of spacers is attributed to phage/plasmid population diversity and retention of old spacers, despite rapid evolution of the targeted phage/plasmid genome regions (proto-spacers). The oldest spacers (spacers found at the trailer end) are conserved for at least 5 years, and 12% of these retain perfect or near-perfect matches to proto-spacer targets. The majority of proto-spacer regions contain an AAG proto-spacer adjacent motif (PAM). Spacers throughout the locus target the same phage population (AMDV1), but there are blocks of consecutive spacers without AMDV1 target sequences. Results suggest long-term coexistence of Leptospirillum with AMDV1 and periods when AMDV1 was less dominant. Metagenomics can be applied to millions of cells in a single sample to provide an extremely large spacer inventory, allow identification of phage/plasmids and enable analysis of previous phage/plasmid exposure. Thus, this approach can provide insights into prior bacterial environment and genetic interplay between hosts and their viruses.

| S-EPMC4796926 | biostudies-literature

CRISPR/Cas9 editing reveals novel mechanisms of clustered microRNA regulation and function.

Project description:MicroRNAs (miRNAs) are important regulators of diverse physiological and pathophysiological processes. MiRNA families and clusters are two key features in miRNA biology. Here we explore the use of CRISPR/Cas9 as a powerful tool to delineate the function and regulation of miRNA families and clusters. We focused on four miRNA clusters composed of miRNA members of the same family, homo-clusters or different families, hetero-clusters. Our results highlight different regulatory mechanisms in miRNA cluster expression. In the case of the miR-497~195 cluster, editing of miR-195 led to a significant decrease in the expression of the other miRNA in the cluster, miR-497a. Although no gene editing was detected in the miR-497a genomic locus, computational simulation revealed alteration in the three dimensional structure of the pri-miR-497~195 that may affect its processing. In cluster miR-143~145 our results imply a feed-forward regulation, although structural changes cannot be ruled out. Furthermore, in the miR-17~92 and miR-106~25 clusters no interdependency in miRNA expression was observed. Our findings suggest that CRISPR/Cas9 is a powerful gene editing tool that can uncover novel mechanisms of clustered miRNA regulation and function.

| S-EPMC5561095 | biostudies-literature

Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function.

Project description:A recent genome-wide screen identified ~300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9Smu) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9Smu with a constitutively active single-guide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (Cas9Spy). We then investigated sgRNA specificity and proto-spacer adjacent motif (PAM) requirements. Interference by sgRNAs did not occur with double or triple base-pair mutations, or if single base-pair mutations were in the 3' end of the sgRNA. Bioinformatic analysis of >450 S. mutans genomes allied with in vivo assays revealed a similar PAM recognition sequence as Cas9Spy. Next, we created a comprehensive library of sgRNA plasmids that were directed at essential and growth-supporting genes. We discovered growth defects for 77% of the CRISPRi strains expressing sgRNAs. Phenotypes of CRISPRi strains, across several biological pathways, were assessed using fluorescence microscopy. A variety of cell structure anomalies were observed, including segregational instability of the chromosome, enlarged cells, and ovococci-to-rod shape transitions. CRISPRi was also employed to observe how silencing of cell wall glycopolysaccharide biosynthesis (rhamnose-glucose polysaccharide, RGP) affected both cell division and pathogenesis in a wax worm model. The CRISPRi tool and sgRNA library are valuable resources for characterizing essential genes in S. mutans, some of which could prove to be promising therapeutic targets.

| S-EPMC7082069 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data