Browse
Submit Data
Databases
API
Help

Dataset Information

26 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

CROP: a CRISPR/Cas9 guide selection program based on mapping guide variants.

ABSTRACT: The off-target effect, in which DNA cleavage was conducted outside the targeted region, is a major problem which limits the applications of CRISPR/Cas9 genome editing system. CRISPR Off-target Predictor (CROP) is standalone program developed to address this problem by predicting off-target propensity of guide RNAs and thereby allowing the user to select the optimum guides. The approach used by CROP involves generating substitution, deletion and insertion combinations which are then mapped into the reference genome. Based on these mapped variants, scoring and alignment are conducted and then reported as a table comprising the off-target propensity of all guide RNAs from a given gene sequence. The Python script for this program is freely available from: https://github.com/vaprilyanto/crop .

SUBMITTER: Aprilyanto V

PROVIDER: S-EPMC7811000 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

CROP: a CRISPR/Cas9 guide selection program based on mapping guide variants.

Aprilyanto Victor V Aditama Redi R Tanjung Zulfikar Achmad ZA Utomo Condro C Liwang Tony T

Scientific reports 20210115 1

The off-target effect, in which DNA cleavage was conducted outside the targeted region, is a major problem which limits the applications of CRISPR/Cas9 genome editing system. CRISPR Off-target Predictor (CROP) is standalone program developed to address this problem by predicting off-target propensity of guide RNAs and thereby allowing the user to select the optimum guides. The approach used by CROP involves generating substitution, deletion and insertion combinations which are then mapped into t ...[more]

PMID: 33452424

Similar Datasets

Rational Selection of CRISPR-Cas9 Guide RNAs for Homology-Directed Genome Editing.

Project description:Homology-directed repair (HDR) of a DNA break allows copying of genetic material from an exogenous DNA template and is frequently exploited in CRISPR-Cas9 genome editing. However, HDR is in competition with other DNA repair pathways, including non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ), and the efficiency of HDR outcomes is not predictable. Consequently, to optimize HDR editing, panels of CRISPR-Cas9 guide RNAs (gRNAs) and matched homology templates must be evaluated. We report here that CRISPR-Cas9 indel signatures can instead be used to identify gRNAs that maximize HDR outcomes. Specifically, we show that the frequency of deletions resulting from MMEJ repair, characterized as deletions greater than or equal to 3 bp, better predicts HDR frequency than consideration of total indel frequency. We further demonstrate that tools that predict gRNA indel signatures can be repurposed to identify gRNAs to promote HDR. Finally, by comparing indels generated by S. aureus and S. pyogenes Cas9 targeted to the same site, we add to the growing body of data that the targeted DNA sequence is a major factor governing genome editing outcomes.

| S-EPMC7934447 | biostudies-literature

Optimized CRISPR guide RNA design for two high-fidelity Cas9 variants by deep learning.

Project description:Highly specific Cas9 nucleases derived from SpCas9 are valuable tools for genome editing, but their wide applications are hampered by a lack of knowledge governing guide RNA (gRNA) activity. Here, we perform a genome-scale screen to measure gRNA activity for two highly specific SpCas9 variants (eSpCas9(1.1) and SpCas9-HF1) and wild-type SpCas9 (WT-SpCas9) in human cells, and obtain indel rates of over 50,000 gRNAs for each nuclease, covering ~20,000 genes. We evaluate the contribution of 1,031 features to gRNA activity and develope models for activity prediction. Our data reveals that a combination of RNN with important biological features outperforms other models for activity prediction. We further demonstrate that our model outperforms other popular gRNA design tools. Finally, we develop an online design tool DeepHF for the three Cas9 nucleases. The database, as well as the designer tool, is freely accessible via a web server, http://www.DeepHF.com/ .

| S-EPMC6753114 | biostudies-literature

Selection of highly efficient sgRNAs for CRISPR/Cas9-based plant genome editing.

Project description:The CRISPR/Cas9-sgRNA system has been developed to mediate genome editing and become a powerful tool for biological research. Employing the CRISPR/Cas9-sgRNA system for genome editing and manipulation has accelerated research and expanded researchers' ability to generate genetic models. However, the method evaluating the efficiency of sgRNAs is lacking in plants. Based on the nucleotide compositions and secondary structures of sgRNAs which have been experimentally validated in plants, we instituted criteria to design efficient sgRNAs. To facilitate the assembly of multiple sgRNA cassettes, we also developed a new strategy to rapidly construct CRISPR/Cas9-sgRNA system for multiplex editing in plants. In theory, up to ten single guide RNA (sgRNA) cassettes can be simultaneously assembled into the final binary vectors. As a proof of concept, 21 sgRNAs complying with the criteria were designed and the corresponding Cas9/sgRNAs expression vectors were constructed. Sequencing analysis of transgenic rice plants suggested that 82% of the desired target sites were edited with deletion, insertion, substitution, and inversion, displaying high editing efficiency. This work provides a convenient approach to select efficient sgRNAs for target editing.

| S-EPMC4759811 | biostudies-other

A systematic genome-wide mapping of oncogenic mutation selection during CRISPR-Cas9 genome editing.

Project description:Recent studies have reported that genome editing by CRISPR-Cas9 induces a DNA damage response mediated by p53 in primary cells hampering their growth. This could lead to a selection of cells with pre-existing p53 mutations. In this study, employing an integrated computational and experimental framework, we systematically investigated the possibility of selection of additional cancer driver mutations during CRISPR-Cas9 gene editing. We first confirm the previous findings of the selection for pre-existing p53 mutations by CRISPR-Cas9. We next demonstrate that similar to p53, wildtype KRAS may also hamper the growth of Cas9-edited cells, potentially conferring a selective advantage to pre-existing KRAS-mutant cells. These selective effects are widespread, extending across cell-types and methods of CRISPR-Cas9 delivery and the strength of selection depends on the sgRNA sequence and the gene being edited. The selection for pre-existing p53 or KRAS mutations may confound CRISPR-Cas9 screens in cancer cells and more importantly, calls for monitoring patients undergoing CRISPR-Cas9-based editing for clinical therapeutics for pre-existing p53 and KRAS mutations.

| S-EPMC8586238 | biostudies-literature

WU-CRISPR: characteristics of functional guide RNAs for the CRISPR/Cas9 system.

Project description:The CRISPR/Cas9 system has been rapidly adopted for genome editing. However, one major issue with this system is the lack of robust bioinformatics tools for design of single guide RNA (sgRNA), which determines the efficacy and specificity of genome editing. To address this pressing need, we analyze CRISPR RNA-seq data and identify many novel features that are characteristic of highly potent sgRNAs. These features are used to develop a bioinformatics tool for genome-wide design of sgRNAs with improved efficiency. These sgRNAs as well as the design tool are freely accessible via a web server, WU-CRISPR ( http://crispr.wustl.edu ).

| S-EPMC4629399 | biostudies-literature

A benchmark of computational CRISPR-Cas9 guide design methods.

Project description:The popularity of CRISPR-based gene editing has resulted in an abundance of tools to design CRISPR-Cas9 guides. This is also driven by the fact that designing highly specific and efficient guides is a crucial, but not trivial, task in using CRISPR for gene editing. Here, we thoroughly analyse the performance of 18 design tools. They are evaluated based on runtime performance, compute requirements, and guides generated. To achieve this, we implemented a method for auditing system resources while a given tool executes, and tested each tool on datasets of increasing size, derived from the mouse genome. We found that only five tools had a computational performance that would allow them to analyse an entire genome in a reasonable time, and without exhausting computing resources. There was wide variation in the guides identified, with some tools reporting every possible guide while others filtered for predicted efficiency. Some tools also failed to exclude guides that would target multiple positions in the genome. We also considered two collections with over a thousand guides each, for which experimental data is available. There is a lot of variation in performance between the datasets, but the relative order of the tools is partially conserved. Importantly, the most striking result is a lack of consensus between the tools. Our results show that CRISPR-Cas9 guide design tools need further work in order to achieve rapid whole-genome analysis and that improvements in guide design will likely require combining multiple approaches.

| S-EPMC6738662 | biostudies-literature

CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses.

Project description:A key goal for cell biological analyses is to assess the phenotypes that result from eliminating a target gene. Since the early 1990s, the predominant strategy utilized in human tissue culture cells has been RNA interference (RNAi)-mediated protein depletion. However, RNAi suffers well-documented off-target effects as well as incomplete and reversible protein depletion. The implementation of CRISPR/Cas9-based DNA cleavage has revolutionized the capacity to conduct functional studies in human cells. However, this approach is still underutilized for conducting visual phenotypic analyses, particularly for essential genes that require conditional strategies to eliminate their gene products. Optimizing this strategy requires effective and streamlined approaches to introduce the Cas9 guide RNA into target cells. Here we assess the efficacy of synthetic guide RNA transfection to eliminate gene products for cell biological studies. On the basis of three representative gene targets (KIF11, CENPN, and RELA), we demonstrate that transfection of synthetic single guide RNA (sgRNA) and CRISPR RNA (crRNA) guides works comparably for protein depletion as cell lines stably expressing lentiviral-delivered RNA guides. We additionally demonstrate that synthetic sgRNAs can be introduced by reverse transfection on an array. Together, these strategies provide a robust, flexible, and scalable approach for conducting functional studies in human cells.

| S-EPMC6233062 | biostudies-literature

A geminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing.

Project description:CRISPR/Cas has emerged as potent genome editing technology and has successfully been applied in many organisms, including several plant species. However, delivery of genome editing reagents remains a challenge in plants. Here, we report a virus-based guide RNA (gRNA) delivery system for CRISPR/Cas9 mediated plant genome editing (VIGE) that can be used to precisely target genome locations and cause mutations. VIGE is performed by using a modified Cabbage Leaf Curl virus (CaLCuV) vector to express gRNAs in stable transgenic plants expressing Cas9. DNA sequencing confirmed VIGE of endogenous NbPDS3 and NbIspH genes in non-inoculated leaves because CaLCuV can infect plants systemically. Moreover, VIGE of NbPDS3 and NbIspH in newly developed leaves caused photo-bleached phenotype. These results demonstrate that geminivirus-based VIGE could be a powerful tool in plant genome editing.

| S-EPMC4598821 | biostudies-literature

WheatCRISPR: a web-based guide RNA design tool for CRISPR/Cas9-mediated genome editing in wheat.

Project description:BACKGROUND:CRISPR/Cas9 gene editing has become a revolutionary technique for crop improvement as it can facilitate fast and efficient genetic changes without the retention of transgene components in the final plant line. Lack of robust bioinformatics tools to facilitate the design of highly specific functional guide RNAs (gRNAs) and prediction of off-target sites in wheat is currently an obstacle to effective application of CRISPR technology to wheat improvement. DESCRIPTION:We have developed a web-based bioinformatics tool to design specific gRNAs for genome editing and transcriptional regulation of gene expression in wheat. A collaborative study between the Broad Institute and Microsoft Research used large-scale empirical evidence to devise algorithms (Doech et al., 2016, Nature Biotechnology 34, 184-191) for predicting the on-target activity and off-target potential of CRISPR/SpCas9 (Streptococcus pyogenes Cas9). We applied these prediction models to determine on-target specificity and potential off-target activity for individual gRNAs targeting specific loci in the wheat genome. The genome-wide gRNA mappings and the corresponding Doench scores predictive of the on-target and off-target activities were used to create a gRNA database which was used as a data source for the web application termed WheatCRISPR. CONCLUSION:The WheatCRISPR tool allows researchers to browse all possible gRNAs targeting a gene or sequence of interest and select effective gRNAs based on their predicted high on-target and low off-target activity scores, as well as other characteristics such as position within the targeted gene. It is publicly available at https://crispr.bioinfo.nrc.ca/WheatCrispr/ .

| S-EPMC6836449 | biostudies-literature

Key sequence features of CRISPR RNA for dual-guide CRISPR-Cas9 ribonucleoprotein complexes assembled with wild-type or HiFi Cas9.

Project description:Specific sequence features of the protospacer and protospacer-adjacent motif (PAM) are critical for efficient cleavage by CRISPR-Cas9, but current knowledge is largely derived from single-guide RNA (sgRNA) systems assessed in cultured cells. In this study, we sought to determine gRNA sequence features of a more native CRISPR-Cas9 ribonucleoprotein (RNP) complex with dual-guide RNAs (dgRNAs) composed of crRNA and tracrRNA, which has been used increasingly in recent CRISPR-Cas9 applications, particularly in zebrafish. Using both wild-type and HiFi SpCas9, we determined on-target cleavage efficiencies of 51 crRNAs in zebrafish embryos by assessing indel occurrence. Statistical analysis of these data identified novel position-specific mononucleotide features relevant to cleavage efficiencies throughout the protospacer sequence that may be unique to CRISPR-Cas9 RNPs pre-assembled with perfectly matched gRNAs. Overall features for wild-type Cas9 resembled those for HiFi Cas9, but specific differences were also observed. Mutational analysis of mononucleotide features confirmed their relevance to cleavage efficiencies. Moreover, the mononucleotide feature-based score, CRISPR-kp, correlated well with efficiencies of gRNAs reported in previous zebrafish RNP injection experiments, as well as independently tested crRNAs only in RNP format, but not with Cas9 mRNA co-injection. These findings will facilitate design of gRNA/crRNAs in genome editing applications, especially when using pre-assembled RNPs.

| S-EPMC8934663 | 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