Browse
Submit Data
Databases
API
Help

Dataset Information

29 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

CRISPR RNA-guided autonomous delivery of Cas9.

ABSTRACT: Cas9 is an endonuclease that can be programed to autonomously deliver diverse effectors to specified genetic addresses. High-resolution structures of this protein and its associated CRISPR RNA guide explain the molecular mechanisms of CRISPR-RNA-guided DNA recognition and provide a molecular blueprint that has facilitated structure-guided functional remodeling. Here we retrace events that led from early efforts to understand the central role of Cas9 in CRISPR-mediated adaptive immunity to contemporary efforts aimed at developing and deploying this enzyme for programmable genetic editing.

SUBMITTER: Wilkinson RA

PROVIDER: S-EPMC7703833 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

CRISPR RNA-guided autonomous delivery of Cas9.

Wilkinson Royce A RA Martin Coleman C Nemudryi Artem A AA Wiedenheft Blake B

Nature structural & molecular biology 20181231 1

Cas9 is an endonuclease that can be programed to autonomously deliver diverse effectors to specified genetic addresses. High-resolution structures of this protein and its associated CRISPR RNA guide explain the molecular mechanisms of CRISPR-RNA-guided DNA recognition and provide a molecular blueprint that has facilitated structure-guided functional remodeling. Here we retrace events that led from early efforts to understand the central role of Cas9 in CRISPR-mediated adaptive immunity to contem ...[more]

PMID: 30598555

Similar Datasets

Synthetic CRISPR RNA-Cas9-guided genome editing in human cells.

Project description:Genome editing with the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nuclease system is a powerful technology for manipulating genomes, including introduction of gene disruptions or corrections. Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA), which in combination with unmodified transactivating crRNA (tracrRNA) is shown to functionally replace the natural guide RNA in the CRISPR-Cas9 nuclease system and to mediate efficient genome editing in human cells. Incorporation of rational chemical modifications known to protect against nuclease digestion and stabilize RNA-RNA interactions in the tracrRNA hybridization region of CRISPR RNA (crRNA) yields a scrRNA with enhanced activity compared with the unmodified crRNA and comparable gene disruption activity to the previously published single guide RNA. Taken together, these findings provide a platform for therapeutic applications, especially for nervous system disease, using successive application of cell-permeable, synthetic CRISPR RNAs to activate and then silence Cas9 nuclease activity.

| S-EPMC4697396 | biostudies-other

Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease.

Project description:We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.

| S-EPMC3730909 | biostudies-other

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.

Project description:The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

| S-EPMC4106473 | biostudies-literature

RNA-guided gene activation by CRISPR-Cas9-based transcription factors.

Project description:Technologies for engineering synthetic transcription factors have enabled many advances in medical and scientific research. In contrast to existing methods based on engineering of DNA-binding proteins, we created a Cas9-based transactivator that is targeted to DNA sequences by guide RNA molecules. Coexpression of this transactivator and combinations of guide RNAs in human cells induced specific expression of endogenous target genes, demonstrating a simple and versatile approach for RNA-guided gene activation.

| S-EPMC3911785 | biostudies-literature

Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.

Project description:Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity.

| S-EPMC3856256 | biostudies-literature

Editor's cut: DNA cleavage by CRISPR RNA-guided nucleases Cas9 and Cas12a.

Project description:Discovered as an adaptive immune system of prokaryotes, CRISPR-Cas provides many promising applications. DNA-cleaving Cas enzymes like Cas9 and Cas12a, are of great interest for genome editing. The specificity of these DNA nucleases is determined by RNA guides, providing great targeting adaptability. Besides this general method of programmable DNA cleavage, these nucleases have different biochemical characteristics, that can be exploited for different applications. Although Cas nucleases are highly promising, some room for improvement remains. New developments and discoveries like base editing, prime editing, and CRISPR-associated transposons might address some of these challenges.

| S-EPMC7054755 | biostudies-literature

RNA-Guided CRISPR-Cas9 System-Mediated Engineering of Acute Myeloid Leukemia Mutations.

Project description:Current acute myeloid leukemia (AML) disease models face severe limitations because most of them induce un-physiological gene expressions that do not represent conditions in AML patients and/or depend on external promoters for regulation of gene expression/repression. Furthermore, many AML models are based on reciprocal chromosomal translocations that only reflect the minority of AML patients, whereas more than 50% of patients have a normal karyotype. The majority of AML, however, is driven by somatic mutations. Thus, identification as well as a detailed molecular and functional characterization of the role of these driver mutations via improved AML models is required for better approaches toward novel targeted therapies. Using the IDH2 R140Q mutation as a model, we present a new effective methodology here using the RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to reproduce or remove AML-associated mutations in or from human leukemic cells, respectively, via introduction of a DNA template at the endogenous gene locus via homologous recombination. Our technology represents a precise way for AML modeling to gain insights into AML development and progression and provides a basis for future therapeutic approaches.

| S-EPMC5363507 | biostudies-literature

RNA-guided transcriptional silencing in vivo with S. aureus CRISPR-Cas9 repressors.

Project description:CRISPR-Cas9 transcriptional repressors have emerged as robust tools for disrupting gene regulation in vitro but have not yet been adapted for systemic delivery in adult animal models. Here we describe a Staphylococcus aureus Cas9-based repressor (dSaCas9KRAB) compatible with adeno-associated viral (AAV) delivery. To evaluate dSaCas9KRAB efficacy for gene silencing in vivo, we silenced transcription of Pcsk9, a regulator of cholesterol levels, in the liver of adult mice. Systemic administration of a dual-vector AAV8 system expressing dSaCas9KRAB and a Pcsk9-targeting guide RNA (gRNA) results in significant reductions of serum Pcsk9 and cholesterol levels. Despite a moderate host response to dSaCas9KRAB expression, Pcsk9 repression is maintained for 24 weeks after a single treatment, demonstrating the potential for long-term gene silencing in post-mitotic tissues with dSaCas9KRAB. In vivo programmable gene silencing enables studies that link gene regulation to complex phenotypes and expands the CRISPR-Cas9 perturbation toolbox for basic research and gene therapy applications.

| S-EPMC5920046 | biostudies-literature

Adenoviral vector delivery of RNA-guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse array of human cells.

Project description:CRISPR/Cas9-derived RNA-guided nucleases (RGNs) are DNA targeting systems, which are rapidly being harnessed for gene regulation and gene editing purposes in model organisms and cell lines. As bona fide gene delivery vehicles, viral vectors may be particularly fit to broaden the applicability of RGNs to other cell types including dividing and quiescent primary cells. Here, the suitability of adenoviral vectors (AdVs) for delivering RGN components into various cell types is investigated. We demonstrate that AdVs, namely second-generation fiber-modified AdVs encoding Cas9 or single guide RNA (gRNA) molecules addressing the Cas9 nuclease to the AAVS1 "safe harbor" locus or to a recombinant model allele can be produced to high-titers (up to 20 × 10(10) transducing units/ml). Importantly, AdV-mediated transduction of gRNA:Cas9 ribonucleoprotein complexes into transformed and non-transformed cells yields rates of targeted mutagenesis similar to or approaching those achieved by isogenic AdVs encoding TALENs targeting the same AAVS1 chromosomal region. RGN-induced gene disruption frequencies in the various cell types ranged from 18% to 65%. We conclude that AdVs constitute a valuable platform for introducing RGNs into human somatic cells regardless of their transformation status. This approach should aid investigating the potential and limitations of RGNs in numerous experimental settings.

| S-EPMC4037712 | biostudies-literature

RNA-Guided Human Gene Activation by Cas9/CRISPR-Based Engineered Transcription Factors

Project description:Synthetic transcription factors can be applied in many areas of biotechnology, medicine, and basic research. In contrast to current methods based on engineering new DNA-binding proteins, we show that Cas9 fused to a transcriptional activation domain can be targeted by combinations of guide RNA molecules to induce the expression of endogenous human genes. This simple approach for targeted gene activation circumvents the need for engineering new proteins and will enable widespread synthetic gene regulation. HEK293T cells were transfected with plasmid expressing Cas9-VP64 fusion protein and a guide RNA. As a control, empty guide RNA was transfected. Gene expression was then measured using mRNA-seq, and differential expression calculated using DESeq. All experiments were performed in biological duplicates or triplicates.

2013-07-25 | E-GEOD-47114 | biostudies-arrayexpress

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