Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease.

ABSTRACT: We have developed a CRISPR-based method that uses catalytically active Cas9 and distinct single guide (sgRNA) constructs to knock out and activate different genes in the same cell. These sgRNAs, with 14- to 15-bp target sequences and MS2 binding loops, can activate gene expression using an active Streptococcus pyogenes Cas9 nuclease, without inducing double-stranded breaks. We use these 'dead RNAs' to perform orthogonal gene knockout and transcriptional activation in human cells.

SUBMITTER: Dahlman JE

PROVIDER: S-EPMC4747789 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease.

Dahlman James E JE Abudayyeh Omar O OO Joung Julia J Gootenberg Jonathan S JS Zhang Feng F Konermann Silvana S

Nature biotechnology 20151101 11

We have developed a CRISPR-based method that uses catalytically active Cas9 and distinct single guide (sgRNA) constructs to knock out and activate different genes in the same cell. These sgRNAs, with 14- to 15-bp target sequences and MS2 binding loops, can activate gene expression using an active Streptococcus pyogenes Cas9 nuclease, without inducing double-stranded breaks. We use these 'dead RNAs' to perform orthogonal gene knockout and transcriptional activation in human cells. ...[more]

PMID: 26436575

Similar Datasets

Efficient generation of FVII gene knockout mice using CRISPR/Cas9 nuclease and truncated guided RNAs.

Project description:We investigated the effects of 5'-end truncated CRISPR RNA-guided Cas9 nuclease (tru-RGN, 17/18 nucleotides) on genome editing capability in NIH/3T3 cells, and its efficiencies on generating Factor VII (FVII) gene-knockout (KO) mice. In cultured cells, RGNs on-target editing activity had been varied when gRNAs was truncated, higher at Site Two (tF7-2 vs. F7-2, 49.5 vs. 30.1%) while lower in other two sites (Site One, tF7-1 vs.F7-1, 12.1 vs. 23.6%; Site Three, tF7-3 vs.F7-3, 7.7 vs 10.9%) (P?<?0.05). Out of 15 predicated off-target sites, tru-RGNs showed significantly decreased frequencies at 5 sites. By microinjecting tru-RGN RNAs into zygotes, FVII KO mice were generated with higher efficiency at Site Two (80.1 vs. 35.8%) and Site One (55.0 vs 3.7%) (P?<?0.05), but not at Site three (39.4 vs 27.8%) (P?>?0.05) when compared with standard RGN controls. Knockout FVII mice demonstrated a delayed prothrombin time and decreased plasma FVII expression. Our study first demonstrates that truncated gRNAs to 18 complementary nucleotides and Cas9 nucleases, can effectively generate FVII gene KO mice with a significantly higher efficiency in a site-dependent manner. In addition, the off-target frequency was much lower in KO mice than in cell lines via RGN expression vector-mediated genome editing.

| S-EPMC4853708 | biostudies-literature

Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification.

Project description:Genome editing by Cas9, which cleaves double-stranded DNA at a sequence programmed by a short single-guide RNA (sgRNA), can result in off-target DNA modification that may be detrimental in some applications. To improve DNA cleavage specificity, we generated fusions of catalytically inactive Cas9 and FokI nuclease (fCas9). DNA cleavage by fCas9 requires association of two fCas9 monomers that simultaneously bind target sites ∼15 or 25 base pairs apart. In human cells, fCas9 modified target DNA sites with >140-fold higher specificity than wild-type Cas9 and with an efficiency similar to that of paired Cas9 'nickases', recently engineered variants that cleave only one DNA strand per monomer. The specificity of fCas9 was at least fourfold higher than that of paired nickases at loci with highly similar off-target sites. Target sites that conform to the substrate requirements of fCas9 occur on average every 34 bp in the human genome, suggesting the versatility of this approach for highly specific genome-wide editing.

| S-EPMC4263420 | biostudies-literature

Catalytically Active Cas9 Mediates Transcriptional Interference to Facilitate Bacterial Virulence.

Project description:In addition to defense against foreign DNA, the CRISPR-Cas9 system of Francisella novicida represses expression of an endogenous immunostimulatory lipoprotein. We investigated the specificity and molecular mechanism of this regulation, demonstrating that Cas9 controls a highly specific regulon of four genes that must be repressed for bacterial virulence. Regulation occurs through a protospacer adjacent motif (PAM)-dependent interaction of Cas9 with its endogenous DNA targets, dependent on a non-canonical small RNA (scaRNA) and tracrRNA. The limited complementarity between scaRNA and the endogenous DNA targets precludes cleavage, highlighting the evolution of scaRNA to repress transcription without lethally targeting the chromosome. We show that scaRNA can be reprogrammed to repress other genes, and with engineered, extended complementarity to an exogenous target, the repurposed scaRNA:tracrRNA-FnoCas9 machinery can also direct DNA cleavage. Natural Cas9 transcriptional interference likely represents a broad paradigm of regulatory functionality, which is potentially critical to the physiology of numerous Cas9-encoding pathogenic and commensal organisms.

| S-EPMC7205310 | biostudies-literature

Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening.

Project description:Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom- or ready-made guide RNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified by the screen, we further describe strategies for confirming the screening phenotype, as well as genetic perturbation, through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 9-15 weeks, followed by 4-5 weeks of validation.

| S-EPMC5526071 | biostudies-literature

Orthogonal Cas9 proteins for RNA-guided gene regulation and editing.

Project description:The Cas9 protein from the Streptococcus pyogenes CRISPR-Cas acquired immune system has been adapted for both RNA-guided genome editing and gene regulation in a variety of organisms, but it can mediate only a single activity at a time within any given cell. Here we characterize a set of fully orthogonal Cas9 proteins and demonstrate their ability to mediate simultaneous and independently targeted gene regulation and editing in bacteria and in human cells. We find that Cas9 orthologs display consistent patterns in their recognition of target sequences, and we identify an unexpectedly versatile Cas9 protein from Neisseria meningitidis. We provide a basal set of orthogonal RNA-guided proteins for controlling biological systems and establish a general methodology for characterizing additional proteins.

| S-EPMC3844869 | biostudies-literature

Site-specific chromosomal gene insertion: Flp recombinase versus Cas9 nuclease.

Project description:Site-specific recombination systems like those based on the Flp recombinase proved themselves as efficient tools for cell line engineering. The recent emergence of designer nucleases, especially RNA guided endonucleases like Cas9, has considerably broadened the available toolbox for applications like targeted transgene insertions. Here we established a recombinase-mediated cassette exchange (RMCE) protocol for the fast and effective, drug-free isolation of recombinant cells. Distinct fluorescent protein patterns identified the recombination status of individual cells. In derivatives of a CHO master cell line the expression of the introduced transgene of interest could be dramatically increased almost 20-fold by subsequent deletion of the fluorescent protein gene that provided the initial isolation principle. The same master cell line was employed in a comparative analysis using CRISPR/Cas9 for transgene integration in identical loci. Even though the overall targeting efficacy was comparable, multi-loci targeting was considerably more effective for Cas9-mediated transgene insertion when compared to RMCE. While Cas9 is inherently more flexible, our results also alert to the risk of aberrant recombination events around the cut site. Together, this study points at the individual strengths in performance of both systems and provides guidance for their appropriate use.

| S-EPMC5736728 | biostudies-literature

Orthogonal Modular Gene Repression in Escherichia coli Using Engineered CRISPR/Cas9.

Project description:The progress in development of synthetic gene circuits has been hindered by the limited repertoire of available transcription factors. Recently, it has been greatly expanded using the CRISPR/Cas9 system. However, this system is limited by its imperfect DNA sequence specificity, leading to potential crosstalk with host genome or circuit components. Furthermore, CRISPR/Cas9-mediated gene regulation is context dependent, affecting the modularity of Cas9-based transcription factors. In this paper we address the problems of specificity and modularity by developing a computational approach for selecting Cas9/gRNA transcription factor/promoter pairs that are maximally orthogonal to each other as well as to the host genome and synthetic circuit components. We validate the method by designing and experimentally testing four orthogonal promoter/repressor pairs in the context of a strong promoter PL from phage lambda. We demonstrate that these promoters can be interfaced by constructing double and triple inverter circuits. To address the problem of modularity we propose and experimentally validate a scheme to predictably incorporate orthogonal CRISPR/Cas9 regulation into a large class of natural promoters.

| S-EPMC5462464 | biostudies-literature

Fusion guide RNAs for orthogonal gene manipulation with Cas9 and Cpf1.

Project description:The bacteria-derived clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are powerful tools for genome engineering. Recently, in addition to Cas protein engineering, the improvement of guide RNAs are also performed, contributing to broadening the research area of CRISPR-Cas9 systems. Here we develop a fusion guide RNA (fgRNA) that functions with both Cas9 and Cpf1 proteins to induce mutations in human cells. Furthermore, we demonstrate that fgRNAs can be used in multiplex genome editing and orthogonal genome manipulation with two types of Cas proteins. Our results show that fgRNAs can be used as a tool for performing multiple gene manipulations.

| S-EPMC5700056 | biostudies-literature

Targeted gene knockout by direct delivery of zinc-finger nuclease proteins.

Project description:Zinc-finger nucleases (ZFNs) are versatile reagents that have redefined genome engineering. Realizing the full potential of this technology requires the development of safe and effective methods for delivering ZFNs into cells. We demonstrate the intrinsic cell-penetrating capabilities of the standard ZFN architecture and show that direct delivery of ZFNs as proteins leads to efficient endogenous gene disruption in various mammalian cell types with minimal off-target effects.

| S-EPMC3424280 | biostudies-literature

Catalytically Enhanced Cas9 through Directed Protein Evolution.

Project description:Guided by the extensive knowledge of CRISPR-Cas9 molecular mechanisms, protein engineering can be an effective method in improving CRISPR-Cas9 toward desired traits different from those of their natural forms. Here, we describe a directed protein evolution method that enables selection of catalytically enhanced CRISPR-Cas9 variants (CECas9) by targeting a shortened protospacer within a toxic gene. We demonstrate the effectiveness of this method with a previously characterized Type II-C Cas9 from Acidothermus cellulolyticus (AceCas9) and show by enzyme kinetics an up to fourfold improvement of the in vitro catalytic efficiency by AceCECas9. We further evolved the more widely used Streptococcus pyogenes Cas9 (SpyCas9) and demonstrated a noticeable improvement in the SpyCECas9-facilitated homology directed repair-based gene insertion in human colon cancer cells.

| S-EPMC8182482 | 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