Browse
Submit Data
Databases
API
Help

Dataset Information

19 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases.

ABSTRACT: High-fidelity SpCas9 variants (eSpCas9 and SpCas9-HF1) have been engineered to reduce off-target effects. We found that changes in guide RNA length induced significant reductions in the editing activities of SpCas9 variants in plant cells. Single guide RNAs harboring precise, perfectly matched 20-nucleotide guide sequences are necessary for high on-target editing activities of eSpCas9 and SpCas9-HF1. Precise 20-nucleotide guide sequences derived from tRNA-sgRNA precursors enable robust on-target editing by these variants with enhanced specificity. Our work reveals an effective way of enhancing the use of the high-fidelity SpCas9 nucleases for efficient and precise genome engineering.

SUBMITTER: Zhang D

PROVIDER: S-EPMC5637269 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases.

Zhang Dingbo D Zhang Huawei H Li Tingdong T Chen Kunling K Qiu Jin-Long JL Gao Caixia C

Genome biology 20171011 1

High-fidelity SpCas9 variants (eSpCas9 and SpCas9-HF1) have been engineered to reduce off-target effects. We found that changes in guide RNA length induced significant reductions in the editing activities of SpCas9 variants in plant cells. Single guide RNAs harboring precise, perfectly matched 20-nucleotide guide sequences are necessary for high on-target editing activities of eSpCas9 and SpCas9-HF1. Precise 20-nucleotide guide sequences derived from tRNA-sgRNA precursors enable robust on-target ...[more]

PMID: 29020979

Similar Datasets

Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage.

Project description:BACKGROUND:The propensity for off-target activity of Streptococcus pyogenes Cas9 (SpCas9) has been considerably decreased by rationally engineered variants with increased fidelity (eSpCas9; SpCas9-HF1). However, a subset of targets still generate considerable off-target effects. To deal specifically with these targets, we generated new "Highly enhanced Fidelity" nuclease variants (HeFSpCas9s) containing mutations from both eSpCas9 and SpCas9-HF1 and examined these improved nuclease variants side by side to decipher the factors that affect their specificities and to determine the optimal nuclease for applications sensitive to off-target effects. RESULTS:These three increased-fidelity nucleases can routinely be used only with perfectly matching 20-nucleotide-long spacers, a matching 5' G extension being more detrimental to their activities than a mismatching one. HeFSpCas9 exhibit substantially improved specificity for those targets for which eSpCas9 and SpCas9-HF1 have higher off-target propensity. The targets can also be ranked by their cleavability and off-target effects manifested by the increased fidelity nucleases. Furthermore, we show that the mutations in these variants may diminish the cleavage, but not the DNA-binding, of SpCas9s. CONCLUSIONS:No single nuclease variant shows generally superior fidelity; instead, for highest specificity cleavage, each target needs to be matched with an appropriate high-fidelity nuclease. We provide here a framework for generating new nuclease variants for targets that currently have no matching optimal nuclease, and offer a simple means for identifying the optimal nuclease for targets in the absence of accurate target-ranking prediction tools.

| S-EPMC6389135 | biostudies-literature

Genome editing by dual nucleases records deep lineage hierarchy with high fidelity.

Project description:Single-cell lineage tracing based on CRISPR/Cas9 gene editing tracks both cell states and genetic lineages at a single-cell resolution. We developed DuTracer, a single-cell lineage tracing method, to avoid rapid target exhaustion and barcode information dropout. Firstly, we introduced this system into HEK293T cells and initiated DuTracer for tracing. We collected single-cell transcriptome and barcode amplicon, demonstrating that this system could record more hierarchical information with minimal inter-site deletions. Subsequently, we applied DuTracer to a mouse embryoid body differentiation model to track lineage transitions of different cell types. We revealed cell potency hierarchy during the stochastic differentiation process of mES cells.

2024-07-01 | GSE236936 | GEO

Aptazyme-embedded guide RNAs enable ligand-responsive genome editing and transcriptional activation.

Project description:Programmable sequence-specific genome editing agents such as CRISPR-Cas9 have greatly advanced our ability to manipulate the human genome. Although canonical forms of genome-editing agents and programmable transcriptional regulators are constitutively active, precise temporal and spatial control over genome editing and transcriptional regulation activities would enable the more selective and potentially safer use of these powerful technologies. Here, by incorporating ligand-responsive self-cleaving catalytic RNAs (aptazymes) into guide RNAs, we developed a set of aptazyme-embedded guide RNAs that enable small molecule-controlled nuclease-mediated genome editing and small molecule-controlled base editing, as well as small molecule-dependent transcriptional activation in mammalian cells.

| S-EPMC5493748 | biostudies-literature

Structural insights into a high fidelity variant of SpCas9.

Project description:The RNA-guided endonucleases of the CRISPR-Cas9 system, including the most widely used Cas9 from Streptococcus pyogenes (SpCas9), are becoming a robust genome editing tool in model organisms and hold immense promise for therapeutic applications. Many strategies have been employed to overcome the limitations caused by SpCas9's off-target effects and its stringent requirement for the protospacer adjacent motif (PAM) sequence. However, the structural mechanisms underlying these strategies remain undefined. Here, we present crystal structure of a SpCas9 variant, xCas9 3.7 that has broad PAM compatibility and high DNA targeting specificity, in complex with a single-guide RNA and its double-stranded DNA targets. Structural comparison revealed that salt bridge-stabilized R1335 is critical for the stringent selection of PAM sequence by SpCas9. Unrestricted rotamerization of this residue by the E1219V mutation in xCas9 3.7 lessens the stringency for PAM recognition and allows SpCas9 to recognize multiple PAM sequences as further supported by biochemical data. Compared to those in wild-type (WT) SpCas9, REC2 and REC3 domains in xCas9 3.7 undergo striking conformational changes, leading to reduced contact with DNA substrate. SpCas9 mutants engineered to display less interaction with DNA and have conformationally more flexible REC2 and REC3 domains display enhanced specificity for DNA substrates in both biochemical and cellular assays. Taken together, our findings reveal the structural mechanisms underlying the broadened PAM compatibility and high DNA fidelity of xCas9 3.7, which can assist rational engineering of more efficient SpCas9 variants and probably other Cas9 orthologs.

| S-EPMC6460432 | biostudies-literature

Robust RNA editing via recruitment of endogenous ADARs using circular guide RNAs

Project description:We engineered circular ADAR recruiting guide RNAs (cadRNAs) that efficiently recruit endogenous ADARs to edit specific sites on target RNA

2021-11-15 | GSE164956 | GEO

Self-cleaving guide RNAs enable pharmacological selection of precise gene editing events in vivo

Project description:In this study we compared the gene expression of mice that had received the rAAV-GR-Cypor scgRNA and rAAV-spCas9 with and without APAP selection. APAP selected mice were also compared to mice which had received the rAAV-GR-Hpd miRNA.

2022-10-25 | GSE216550 | GEO

Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9.

Project description:Well-developed genetic tools for thermophilic microorganisms are scarce, despite their industrial and scientific relevance. Whereas highly efficient CRISPR/Cas9-based genome editing is on the rise in prokaryotes, it has never been employed in a thermophile. Here, we apply Streptococcus pyogenes Cas9 (spCas9)-based genome editing to a moderate thermophile, i.e., Bacillus smithii, including a gene deletion, gene knockout via insertion of premature stop codons, and gene insertion. We show that spCas9 is inactive in vivo above 42 °C, and we employ the wide temperature growth range of B. smithii as an induction system for spCas9 expression. Homologous recombination with plasmid-borne editing templates is performed at 45-55 °C, when spCas9 is inactive. Subsequent transfer to 37 °C allows for counterselection through production of active spCas9, which introduces lethal double-stranded DNA breaks to the nonedited cells. The developed method takes 4 days with 90, 100, and 20% efficiencies for gene deletion, knockout, and insertion, respectively. The major advantage of our system is the limited requirement for genetic parts: only one plasmid, one selectable marker, and a promoter are needed, and the promoter does not need to be inducible or well-characterized. Hence, it can be easily applied for genome editing purposes in both mesophilic and thermophilic nonmodel organisms with a limited genetic toolbox and ability to grow at, or tolerate, temperatures of 37 and at or above 42 °C.

| S-EPMC5440800 | biostudies-literature

CLUSTER guide RNAs enable precise and efficient RNA editing with endogenous ADAR enzymes in vivo

Project description:RNA base editing represents a promising alternative for genome editing. Recent approaches harness the endogenous RNA editing enzyme ADAR to circumvent problems related to the ectopic expression of an editing enzyme, but they suffer from sequence restriction, lack of efficiency, and bystander editing. Here, we present in‐silico optimized CLUSTER guide RNAs, which bind their target mRNAs in a multivalent fashion and thereby enable editing with unprecedented precision as shown by next generation sequencing. CLUSTER guide RNAs can be genetically encoded and manufactured into viruses to work in various cell lines. They achieve on‐target editing on endogenous transcripts like GUSB and NUP43 with yields up to 45% without bystander editing and have been shown to recruit endogenous ADAR in vivo. The CLUSTER approach tremendously enlarges the sequence space available for guide RNA design and opens new avenues for drug development in the field of RNA base editing.

2021-09-20 | GSE184244 | GEO

Monomeric site-specific nucleases for genome editing.

Project description:Targeted manipulation of complex genomes often requires the introduction of a double-strand break at defined locations by site-specific DNA endonucleases. Here, we describe a monomeric nuclease domain derived from GIY-YIG homing endonucleases for genome-editing applications. Fusion of the GIY-YIG nuclease domain to three-member zinc-finger DNA binding domains generated chimeric GIY-zinc finger endonucleases (GIY-ZFEs). Significantly, the I-TevI-derived fusions (Tev-ZFEs) function in vitro as monomers to introduce a double-strand break, and discriminate in vitro and in bacterial and yeast assays against substrates lacking a preferred 5'-CNNNG-3' cleavage motif. The Tev-ZFEs function to induce recombination in a yeast-based assay with activity on par with a homodimeric Zif268 zinc-finger nuclease. We also fused the I-TevI nuclease domain to a catalytically inactive LADGLIDADG homing endonuclease (LHE) scaffold. The monomeric Tev-LHEs are active in vivo and similarly discriminate against substrates lacking the 5'-CNNNG-3' motif. The monomeric Tev-ZFEs and Tev-LHEs are distinct from the FokI-derived zinc-finger nuclease and TAL effector nuclease platforms as the GIY-YIG domain alleviates the requirement to design two nuclease fusions to target a given sequence, highlighting the diversity of nuclease domains with distinctive biochemical properties suitable for genome-editing applications.

| S-EPMC3361397 | biostudies-literature

Carboxylated branched poly(?-amino ester) nanoparticles enable robust cytosolic protein delivery and CRISPR-Cas9 gene editing.

Project description:Efficient cytosolic protein delivery is necessary to fully realize the potential of protein therapeutics. Current methods of protein delivery often suffer from low serum tolerance and limited in vivo efficacy. Here, we report the synthesis and validation of a previously unreported class of carboxylated branched poly(?-amino ester)s that can self-assemble into nanoparticles for efficient intracellular delivery of a variety of different proteins. In vitro, nanoparticles enabled rapid cellular uptake, efficient endosomal escape, and functional cytosolic protein release into cells in media containing 10% serum. Moreover, nanoparticles encapsulating CRISPR-Cas9 ribonucleoproteins (RNPs) induced robust levels of gene knock-in (4%) and gene knockout (>75%) in several cell types. A single intracranial administration of nanoparticles delivering a low RNP dose (3.5 pmol) induced robust gene editing in mice bearing engineered orthotopic murine glioma tumors. This self-assembled polymeric nanocarrier system enables a versatile protein delivery and gene editing platform for biological research and therapeutic applications.

| S-EPMC6897553 | 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