Browse
Submit Data
Databases
API
Help

Dataset Information

20 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The Current State and Future of CRISPR-Cas9 gRNA Design Tools.

ABSTRACT: Recent years have seen the development of computational tools to assist researchers in performing CRISPR-Cas9 experiment optimally. More specifically, these tools aim to maximize on-target activity (guide efficiency) while also minimizing potential off-target effects (guide specificity) by analyzing the features of the target site. Nonetheless, currently available tools cannot robustly predict experimental success as prediction accuracy depends on the approximations of the underlying model and how closely the experimental setup matches the data the model was trained on. Here, we present an overview of the available computational tools, their current limitations and future considerations. We discuss new trends around personalized health by taking genomic variants into account when predicting target sites as well as discussing other governing factors that can improve prediction accuracy.

SUBMITTER: Wilson LOW

PROVIDER: S-EPMC6052051 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

The Current State and Future of CRISPR-Cas9 gRNA Design Tools.

Wilson Laurence O W LOW O'Brien Aidan R AR Bauer Denis C DC Bauer Denis C DC

Frontiers in pharmacology 20180712

Recent years have seen the development of computational tools to assist researchers in performing CRISPR-Cas9 experiment optimally. More specifically, these tools aim to maximize on-target activity (guide efficiency) while also minimizing potential off-target effects (guide specificity) by analyzing the features of the target site. Nonetheless, currently available tools cannot robustly predict experimental success as prediction accuracy depends on the approximations of the underlying model and h ...[more]

PMID: 30050439

Similar Datasets

CRISPR/Cas9 Landscape: Current State and Future Perspectives

Project description: Not available

| S-EPMC10671331 | biostudies-literature

CRISPR-Cas9 gRNA efficiency prediction: an overview of predictive tools and the role of deep learning.

Project description:The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has become a successful and promising technology for gene-editing. To facilitate its effective application, various computational tools have been developed. These tools can assist researchers in the guide RNA (gRNA) design process by predicting cleavage efficiency and specificity and excluding undesirable targets. However, while many tools are available, assessment of their application scenarios and performance benchmarks are limited. Moreover, new deep learning tools have been explored lately for gRNA efficiency prediction, but have not been systematically evaluated. Here, we discuss the approaches that pertain to the on-target activity problem, focusing mainly on the features and computational methods they utilize. Furthermore, we evaluate these tools on independent datasets and give some suggestions for their usage. We conclude with some challenges and perspectives about future directions for CRISPR-Cas9 guide design.

| S-EPMC9023298 | biostudies-literature

An overview and metanalysis of machine and deep learning-based CRISPR gRNA design tools.

Project description:The CRISPR-Cas9 system has become the most promising and versatile tool for genetic manipulation applications. Albeit the technology has been broadly adopted by both academic and pharmaceutic societies, the activity (on-target) and specificity (off-target) of CRISPR-Cas9 are decisive factors for any application of the technology. Several in silico gRNA activity and specificity predicting models and web tools have been developed, making it much more convenient and precise for conducting CRISPR gene editing studies. In this review, we present an overview and comparative analysis of machine and deep learning (MDL)-based algorithms, which are believed to be the most effective and reliable methods for the prediction of CRISPR gRNA on- and off-target activities. As an increasing number of sequence features and characteristics are discovered and are incorporated into the MDL models, the prediction outcome is getting closer to experimental observations. We also introduced the basic principle of CRISPR activity and specificity and summarized the challenges they faced, aiming to facilitate the CRISPR communities to develop more accurate models for applying.

| S-EPMC6948960 | biostudies-literature

Highly multiplexed genome engineering using CRISPR/Cas9 gRNA arrays.

Project description:The CRISPR/Cas9 system is an RNA guided nuclease system that evolved as a mechanism of adaptive immunity in bacteria. This system has been adopted for numerous genome engineering applications in research and recently, therapeutics. The CRISPR/Cas9 system has been largely implemented by delivery of Cas9 as protein, RNA, or plasmid along with a chimeric crRNA-tracrRNA guide RNA (gRNA) under the expression of a pol III promoter, such as U6. Using this approach, multiplex genome engineering has been achieved by delivering several U6-gRNA plasmids targeting multiple loci. However, this approach is limited due to the efficiently of delivering multiple plasmids to a single cell at one time. To augment the capability and accessibility of multiplexed genome engineering, we developed an efficient golden gate based method to assemble gRNAs linked by optimal Csy4 ribonuclease sequences to deliver up to 10 gRNAs as a single gRNA array transcript. Here we report the optimal expression of our guide RNA array under a strong pol II promoter. This system can be implemented alongside the myriad of CRISPR applications, allowing users to model complex biological processes requiring numerous gRNAs.

| S-EPMC6141065 | biostudies-literature

Examination of CRISPR/Cas9 design tools and the effect of target site accessibility on Cas9 activity.

Project description:NEW FINDINGS:What is the topic of this review? In this review, we analyse the performance of recently described tools for CRISPR/Cas9 guide RNA design, in particular, design tools that predict CRISPR/Cas9 activity. What advances does it highlight? Recently, many tools designed to predict CRISPR/Cas9 activity have been reported. However, the majority of these tools lack experimental validation. Our analyses indicate that these tools have poor predictive power. Our preliminary results suggest that target site accessibility should be considered in order to develop better guide RNA design tools with improved predictive power. The recent adaptation of the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system for targeted genome engineering has led to its widespread application in many fields worldwide. In order to gain a better understanding of the design rules of CRISPR/Cas9 systems, several groups have carried out large library-based screens leading to some insight into sequence preferences among highly active target sites. To facilitate CRISPR/Cas9 design, these studies have spawned a plethora of guide RNA (gRNA) design tools with algorithms based solely on direct or indirect sequence features. Here, we demonstrate that the predictive power of these tools is poor, suggesting that sequence features alone cannot accurately inform the cutting efficiency of a particular CRISPR/Cas9 gRNA design. Furthermore, we demonstrate that DNA target site accessibility influences the activity of CRISPR/Cas9. With further optimization, we hypothesize that it will be possible to increase the predictive power of gRNA design tools by including both sequence and target site accessibility metrics.

| S-EPMC7266697 | biostudies-literature

Versatile single-step-assembly CRISPR/Cas9 vectors for dual gRNA expression.

Project description:CRISPR/Cas9 technology enables efficient, rapid and cost-effective targeted genomic modification in a wide variety of cellular contexts including cultured cells. Some applications such as generation of double knock-outs, large deletions and paired-nickase cleavage require simultaneous expression of two gRNAs. Although single plasmids that enable multiplex expression of gRNAs have been developed, these require multiple rounds of cloning and/or PCR for generation of the desired construct. Here, we describe a series of vectors that enable generation of customized dual-gRNA expression constructs via an easy one-step golden gate cloning reaction using two annealed oligonucleotide inserts with different overhangs. Through nucleofection of mouse embryonic stem cells, we demonstrate highly efficient cleavage of the target loci using the dual-guide plasmids, which are available as Cas9-nuclease or Cas9-nickase expression constructs, with or without selection markers. These vectors are a valuable addition to the CRISPR/Cas9 toolbox and will be made available to all interested researchers via the Addgene plasmid repository.

| S-EPMC5718404 | biostudies-literature

gRNA validation for wheat genome editing with the CRISPR-Cas9 system.

Project description:BackgroundThe CRISPR-Cas9 system is a powerful and versatile tool for crop genome editing. However, achieving highly efficient and specific editing in polyploid species can be a challenge. The efficiency and specificity of the CRISPR-Cas9 system depends critically on the gRNA used. Here, we assessed the activities and specificities of seven gRNAs targeting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in hexaploid wheat protoplasts. EPSPS is the biological target of the widely used herbicide glyphosate.ResultsThe seven gRNAs differed substantially in their on-target activities, with mean indel frequencies ranging from 0% to approximately 20%. There was no obvious correlation between experimentally determined and in silico predicted on-target gRNA activity. The presence of a single mismatch within the seed region of the guide sequence greatly reduced but did not abolish gRNA activity, whereas the presence of an additional mismatch, or the absence of a PAM, all but abolished gRNA activity. Large insertions (≥20 bp) of DNA vector-derived sequence were detected at frequencies up to 8.5% of total indels. One of the gRNAs exhibited several properties that make it potentially suitable for the development of non-transgenic glyphosate resistant wheat.ConclusionsWe have established a rapid and reliable method for gRNA validation in hexaploid wheat protoplasts. The method can be used to identify gRNAs that have favourable properties. Our approach is particularly suited to polyploid species, but should be applicable to any plant species amenable to protoplast transformation.

| S-EPMC6829922 | biostudies-literature

Synthetic Circular gRNA Mediated Biological Function of CRISPR-(d)Cas9 System.

Project description:Ever since the gene editing function was discovered in the CRISPR-Cas9 system, numerous applications and utilities were investigated in order to apply this technique to medical use. However, the clinical practice was limited by unsatisfactory efficiency and unacceptable off-target editing. Modifications from different aspects of the Cas9 protein and gRNAs were published that aimed to improve its function in one way or another. Under the inspiration of Jacob L. Litke and Samie R. Jaffrey, we propose a novel gRNA design that could achieve rapid circular gRNA assembly inside the cells. This circular design consists of the gRNA of interested flanked by Twister ribozymes. The function of this circular gRNA was proved in vitro in both CRISPR-dCas9 and CRISPR-Cas9 systems. It presented a remarkable reduction in the off-target rate in accompany with reduced efficiency. With future improvement in its efficiency, this tool broadens our understanding and possibility of the CRISPR application.

| S-EPMC9013764 | biostudies-literature

The gRNA-miRNA-gRNA Ternary Cassette Combining CRISPR/Cas9 with RNAi Approach Strongly Inhibits Hepatitis B Virus Replication.

Project description:The CRISPR/Cas9 system is a novel genome editing technology which has been successfully used to inhibit HBV replication. Here, we described a novel gRNA-microRNA (miRNA)-gRNA ternary cassette driven by a single U6 promoter. With an anti-HBV pri-miR31 mimic integrated between two HBV-specific gRNAs, both gRNAs could be separated from the long transcript of gRNA-miR-HBV-gRNA ternary cassette through Drosha/DGCR8 processing. The results showed that the gRNA-miR-HBV-gRNA ternary cassette could efficiently express two gRNAs and miR-HBV. The optimal length of pri-miRNA flanking sequence in our ternary cassette was determined to be 38 base pairs (bp). Besides, HBV-specific gRNAs and miR-HBV in gRNA-miR-HBV-gRNA ternary cassette could exert a synergistic effect in inhibiting HBV replication and destroying HBV genome in vitro and in vivo. Most importantly, together with RNA interference (RNAi) approach, the HBV-specific gRNAs showed the potent activity on the destruction of HBV covalently closed circular DNA (cccDNA). Since HBV cccDNA is an obstacle for the elimination of chronic HBV infection, the gRNA-miR-HBV-gRNA ternary cassette may be a potential tool for the clearance of HBV cccDNA.

| S-EPMC5566108 | biostudies-literature

Rapid and efficient one-step generation of paired gRNA CRISPR-Cas9 libraries.

Project description:The CRISPR-Cas9 system is a powerful tool to edit eukaryotic genomes that has recently been adapted for functional screens. Several of its applications--including the disruption of genes using Cas9-nickase and the generation of large deletions--require co-expression of two distinct guide RNAs (gRNAs). However, the lack of experimental approaches to generate pools of paired gRNA vectors prevents these applications from being scalable. Here we report a simple, inexpensive, one-step method that allows for the rapid and efficient cloning of gRNA pairs into expression vectors. We show that this method can be used to generate pooled libraries and is therefore suitable for in vivo and in vitro functional screens.

| S-EPMC4544769 | 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