GuideScan software for improved single and paired CRISPR guide RNA design.
Ontology highlight
ABSTRACT:
SUBMITTER: Perez AR
PROVIDER: S-EPMC5607865 | biostudies-literature | 2017 Apr
REPOSITORIES: biostudies-literature
Publications
GuideScan software for improved single and paired CRISPR guide RNA design.
Perez Alexendar R AR Pritykin Yuri Y Vidigal Joana A JA Chhangawala Sagar S Zamparo Lee L Leslie Christina S CS Ventura Andrea A
Nature biotechnology 20170306 4
We present GuideScan software for the design of CRISPR guide RNA libraries that can be used to edit coding and noncoding genomic regions. GuideScan produces high-density sets of guide RNAs (gRNAs) for single- and paired-gRNA genome-wide screens. We also show that the trie data structure of GuideScan enables the design of gRNAs that are more specific than those designed by existing tools. ...[more]
Similar Datasets
Project description:Genetic screens using CRISPR/Cas9 are a powerful method for the functional analysis of genomes.Here we describe CRISPR library designer (CLD), an integrated bioinformatics application for the design of custom single guide RNA (sgRNA) libraries for all organisms with annotated genomes. CLD is suitable for the design of libraries using modified CRISPR enzymes and targeting non-coding regions. To demonstrate its utility, we perform a pooled screen for modulators of the TNF-related apoptosis inducing ligand (TRAIL) pathway using a custom library of 12,471 sgRNAs.CLD predicts a high fraction of functional sgRNAs and is publicly available at https://github.com/boutroslab/cld.
| S-EPMC4807595 | biostudies-literature
Project description:BackgroundCRISPR-Cas systems have expanded the possibilities for gene editing in bacteria and eukaryotes. There are many excellent tools for designing CRISPR-Cas guide RNAs (gRNAs) for model organisms with standard Cas enzymes. GuideMaker is intended as a fast and easy-to-use design tool for challenging projects with (i) non-standard Cas enzymes, (ii) non-model organisms, or (iii) projects that need to design a panel of gRNA for genome-wide screens.FindingsGuideMaker can rapidly design gRNAs for gene targets across the genome using a degenerate protospacer-adjacent motif (PAM) and a genome. The tool applies hierarchical navigable small world graphs to speed up the comparison of guide RNAs and optionally provides on-target and off-target scoring. This allows the user to design effective gRNAs targeting all genes in a typical bacterial genome in ∼1-2 minutes.ConclusionsGuideMaker enables the rapid design of genome-wide gRNA for any CRISPR-Cas enzyme in non-model organisms. While GuideMaker is designed with prokaryotic genomes in mind, it can efficiently process eukaryotic genomes as well. GuideMaker is available as command-line software, a stand-alone web application, and a tool in the CyCverse Discovery Environment. All versions are available under a Creative Commons CC0 1.0 Universal Public Domain Dedication.
| S-EPMC8975720 | biostudies-literature
Project description:CRISPR-Cas9 technology can be used to engineer precise genomic deletions with pairs of single guide RNAs (sgRNAs). This approach has been widely adopted for diverse applications, from disease modelling of individual loci, to parallelized loss-of-function screens of thousands of regulatory elements. However, no solution has been presented for the unique bioinformatic design requirements of CRISPR deletion. We here present CRISPETa, a pipeline for flexible and scalable paired sgRNA design based on an empirical scoring model. Multiple sgRNA pairs are returned for each target, and any number of targets can be analyzed in parallel, making CRISPETa equally useful for focussed or high-throughput studies. Fast run-times are achieved using a pre-computed off-target database. sgRNA pair designs are output in a convenient format for visualisation and oligonucleotide ordering. We present pre-designed, high-coverage library designs for entire classes of protein-coding and non-coding elements in human, mouse, zebrafish, Drosophila melanogaster and Caenorhabditis elegans. In human cells, we reproducibly observe deletion efficiencies of ≥50% for CRISPETa designs targeting an enhancer and exonic fragment of the MALAT1 oncogene. In the latter case, deletion results in production of desired, truncated RNA. CRISPETa will be useful for researchers seeking to harness CRISPR for targeted genomic deletion, in a variety of model organisms, from single-target to high-throughput scales.
| S-EPMC5333799 | biostudies-literature
Project description:Success with genome editing by the RNA-programmed nuclease Cas9 has been limited by the inability to predict effective guide RNAs and DNA target sites. Not all guide RNAs have been successful, and even those that were, varied widely in their efficacy. Here we describe and validate a strategy for Caenorhabditis elegans that reliably achieved a high frequency of genome editing for all targets tested in vivo. The key innovation was to design guide RNAs with a GG motif at the 3' end of their target-specific sequences. All guides designed using this simple principle induced a high frequency of targeted mutagenesis via nonhomologous end joining (NHEJ) and a high frequency of precise DNA integration from exogenous DNA templates via homology-directed repair (HDR). Related guide RNAs having the GG motif shifted by only three nucleotides showed severely reduced or no genome editing. We also combined the 3' GG guide improvement with a co-CRISPR/co-conversion approach. For this co-conversion scheme, animals were only screened for genome editing at designated targets if they exhibited a dominant phenotype caused by Cas9-dependent editing of an unrelated target. Combining the two strategies further enhanced the ease of mutant recovery, thereby providing a powerful means to obtain desired genetic changes in an otherwise unaltered genome.
| S-EPMC4391549 | biostudies-literature
Project description:Specific applications of CRISPR/Cas genome editing systems benefit from chemical modifications of the sgRNA. Herein we describe a versatile and efficient strategy for functionalization of the 3'-end of a sgRNA. An exemplary collection of six chemically modified sgRNAs was prepared containing crosslinkers, a fluorophore and biotin. Modification of the sgRNA 3'-end was broadly tolerated by Streptococcus pyogenes Cas9 in an in?vitro DNA cleavage assay. The 3'-biotinylated sgRNA was used as an affinity reagent to identify IGF2BP1, YB1 and hnRNP?K as sgRNA-binding proteins present in HEK293T cells. Overall, the modification strategy presented here has the potential to expand on current applications of CRISPR/Cas systems.
| S-EPMC7323579 | biostudies-literature
Project description:A major challenge for effective application of CRISPR systems is to accurately predict the single guide RNA (sgRNA) on-target knockout efficacy and off-target profile, which would facilitate the optimized design of sgRNAs with high sensitivity and specificity. Here we present DeepCRISPR, a comprehensive computational platform to unify sgRNA on-target and off-target site prediction into one framework with deep learning, surpassing available state-of-the-art in silico tools. In addition, DeepCRISPR fully automates the identification of sequence and epigenetic features that may affect sgRNA knockout efficacy in a data-driven manner. DeepCRISPR is available at http://www.deepcrispr.net/ .
| S-EPMC6020378 | biostudies-literature
Project description:UnlabelledCRISPRdirect is a simple and functional web server for selecting rational CRISPR/Cas targets from an input sequence. The CRISPR/Cas system is a promising technique for genome engineering which allows target-specific cleavage of genomic DNA guided by Cas9 nuclease in complex with a guide RNA (gRNA), that complementarily binds to a ∼ 20 nt targeted sequence. The target sequence requirements are twofold. First, the 5'-NGG protospacer adjacent motif (PAM) sequence must be located adjacent to the target sequence. Second, the target sequence should be specific within the entire genome in order to avoid off-target editing. CRISPRdirect enables users to easily select rational target sequences with minimized off-target sites by performing exhaustive searches against genomic sequences. The server currently incorporates the genomic sequences of human, mouse, rat, marmoset, pig, chicken, frog, zebrafish, Ciona, fruit fly, silkworm, Caenorhabditis elegans, Arabidopsis, rice, Sorghum and budding yeast.AvailabilityFreely available at http://crispr.dbcls.jp/.Supplementary informationSupplementary data are available at Bioinformatics online.
| S-EPMC4382898 | biostudies-literature
Project description:CRISPR/Cas9 is a promising RNA-guided genome editing technology, which consists of a Cas9 nuclease and a single-guide RNA (sgRNA). So far, a number of sgRNA prediction softwares have been developed. However, they were usually designed for protein-coding genes without considering that long non-coding RNA (lncRNA) genes may have different characteristics. In this study, we first evaluated the performances of a series of known sgRNA-designing tools in the context of both coding and non-coding datasets. Meanwhile, we analyzed the underpinnings of their varied performances on the sgRNA's specificity for lncRNA including nucleic acid sequence, genome location and editing mechanism preference. Furthermore, we introduce a support vector machine-based machine learning algorithm named CRISPRlnc, which aims to model both CRISPR knock-out (CRISPRko) and CRISPR inhibition (CRISPRi) mechanisms to predict the on-target activity of targets. CRISPRlnc combined the paired-sgRNA design and off-target analysis to achieve one-stop design of CRISPR/Cas9 sgRNAs for non-coding genes. Performance comparison on multiple datasets showed that CRISPRlnc was far superior to existing methods for both CRISPRko and CRISPRi mechanisms during the lncRNA-specific sgRNA design. To maximize the availability of CRISPRlnc, we developed a web server (http://predict.crisprlnc.cc) and made it available for download on GitHub.
| S-EPMC10905519 | biostudies-literature
Project description:Rapid and accurate diagnostic tests are fundamental for improving patient outcomes and combating infectious diseases. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas12a-based detection system has emerged as a promising solution for on-site nucleic acid testing. Nonetheless, the effective design of CRISPR RNA (crRNA) for Cas12a-based detection remains challenging and time-consuming. In this study, we propose an enhanced crRNA design system with deep learning for Cas12a-mediated diagnostics, referred to as EasyDesign. This system employs an optimized convolutional neural network (CNN) prediction model, trained on a comprehensive data set comprising 11,496 experimentally validated Cas12a-based detection cases, encompassing a wide spectrum of prevalent pathogens, achieving Spearman's ρ = 0.812. We further assessed the model performance in crRNA design for four pathogens not included in the training data: Monkeypox Virus, Enterovirus 71, Coxsackievirus A16, and Listeria monocytogenes. The results demonstrated superior prediction performance compared to the traditional experiment screening. Furthermore, we have developed an interactive web server (https://crispr.zhejianglab.com/) that integrates EasyDesign with recombinase polymerase amplification (RPA) primer design, enhancing user accessibility. Through this web-based platform, we successfully designed optimal Cas12a crRNAs for six human papillomavirus (HPV) subtypes. Remarkably, all the top five predicted crRNAs for each HPV subtype exhibited robust fluorescent signals in CRISPR assays, thereby suggesting that the platform could effectively facilitate clinical sample testing. In conclusion, EasyDesign offers a rapid and reliable solution for crRNA design in Cas12a-based detection, which could serve as a valuable tool for clinical diagnostics and research applications.
| S-EPMC11316927 | biostudies-literature
Project description:CRISPR-Cas9 screens have been widely adopted to analyze coding-gene functions, but high-throughput screening of non-coding elements using this method is more challenging because indels caused by a single cut in non-coding regions are unlikely to produce a functional knockout. A high-throughput method to produce deletions of non-coding DNA is needed. We report a high-throughput genomic deletion strategy to screen for functional long non-coding RNAs (lncRNAs) that is based on a lentiviral paired-guide RNA (pgRNA) library. Applying our screening method, we identified 51 lncRNAs that can positively or negatively regulate human cancer cell growth. We validated 9 of 51 lncRNA hits using CRISPR-Cas9-mediated genomic deletion, functional rescue, CRISPR activation or inhibition and gene-expression profiling. Our high-throughput pgRNA genome deletion method will enable rapid identification of functional mammalian non-coding elements.
| S-EPMC5592164 | biostudies-literature