CRISPR-Local: a local single-guide RNA (sgRNA) design tool for non-reference plant genomes

2018 ◽  
Vol 35 (14) ◽  
pp. 2501-2503 ◽  
Author(s):  
Jiamin Sun ◽  
Hao Liu ◽  
Jianxiao Liu ◽  
Shikun Cheng ◽  
Yong Peng ◽  
...  
Keyword(s):  
Design Tool ◽  
Supplementary Information ◽  
Guide Rna ◽  
Plant Genomes ◽  
Guide Rnas ◽  
Genome Wide ◽  
Reference Plant ◽  
Sgrna Design ◽  
Computational Resources ◽  
Local Tool

Abstract Summary CRISPR-Local is a high-throughput local tool for designing single-guide RNAs (sgRNAs) in plants and other organisms that factors in genetic variation and is optimized to generate genome-wide sgRNAs. CRISPR-Local outperforms other sgRNA design tools in the following respects: (i) designing sgRNAs suitable for non-reference varieties; (ii) screening for sgRNAs that are capable of simultaneously targeting multiple genes; (iii) saving computational resources by avoiding repeated calculations from multiple submissions and (iv) running offline, with both command-line and graphical user interface modes and the ability to export multiple formats for further batch analysis or visualization. We have applied CRISPR-Local to 71 public plant genomes, using both CRISPR/Cas9 and CRISPR/cpf1 systems. Availability and implementation CRISPR-Local can be freely downloaded from http://crispr.hzau.edu.cn/CRISPR-Local/. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals PAVOOC: designing CRISPR sgRNAs using 3D protein structures and functional domain annotations

2018 ◽  
Vol 35 (13) ◽  
pp. 2309-2310 ◽  
Author(s):  
Moritz Schaefer ◽  
Djork-Arné Clevert ◽  
Bertram Weiss ◽  
Andreas Steffen
Keyword(s):  
Protein Structures ◽  
Design Tool ◽  
Protein Crystal ◽  
Supplementary Information ◽  
Functional Domain ◽  
Web Based ◽  
Homology Directed Repair ◽  
Guide Rnas ◽  
Sgrna Design ◽  
Repair Template

Abstract Summary Single-guide RNAs (sgRNAs) targeting the same gene can significantly vary in terms of efficacy and specificity. PAVOOC (Prediction And Visualization of On- and Off-targets for CRISPR) is a web-based CRISPR sgRNA design tool that employs state of the art machine learning models to prioritize most effective candidate sgRNAs. In contrast to other tools, it maps sgRNAs to functional domains and protein structures and visualizes cut sites on corresponding protein crystal structures. Furthermore, PAVOOC supports homology-directed repair template generation for genome editing experiments and the visualization of the mutated amino acids in 3D. Availability and implementation PAVOOC is available under https://pavooc.me and accessible using modern browsers (Chrome/Chromium recommended). The source code is hosted at github.com/moritzschaefer/pavooc under the MIT License. The backend, including data processing steps, and the frontend are implemented in Python 3 and ReactJS, respectively. All components run in a simple Docker environment. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Generalizable sgRNA design for improved CRISPR/Cas9 editing efficiency

2020 ◽  
Vol 36 (9) ◽  
pp. 2684-2689 ◽  
Author(s):  
Kasidet Hiranniramol ◽  
Yuhao Chen ◽  
Weijun Liu ◽  
Xiaowei Wang
Keyword(s):  
Web Application ◽  
Learning Algorithm ◽  
Structural Features ◽  
Design Tool ◽  
Supplementary Information ◽  
Experimental Conditions ◽  
Guide Rna ◽  
Plasmid Library ◽  
Short Palindromic Repeat ◽  
Sgrna Design

Abstract Motivation The development of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has provided a simple yet powerful system for targeted genome editing. In recent years, this system has been widely used for various gene editing applications. The CRISPR editing efficacy is mainly dependent on the single guide RNA (sgRNA), which guides Cas9 for genome cleavage. While there have been multiple attempts at improving sgRNA design, there is a pressing need for greater sgRNA potency and generalizability across various experimental conditions. Results We employed a unique plasmid library expressed in human cells to quantify the potency of thousands of CRISPR/Cas9 sgRNAs. Differential sequence and structural features among the most and least potent sgRNAs were then used to train a machine learning algorithm for assay design. Comparative analysis indicates that our new algorithm outperforms existing CRISPR/Cas9 sgRNA design tools. Availability and implementation The new sgRNA design tool is freely accessible as a web application, http://crispr.wustl.edu. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals GuideMaker: Software to design CRISPR-Cas guide RNA pools in non-model genomes

2021 ◽  
Author(s):  
Ravin Poudel ◽  
Lidimarie Trujillo Rodriguez ◽  
Christopher R Reisch ◽  
Adam R Rivers
Keyword(s):  
Web Application ◽  
Bacterial Genome ◽  
Small World ◽  
Design Tool ◽  
Model Organisms ◽  
Guide Rna ◽  
Creative Commons ◽  
Guide Rnas ◽  
Genome Wide ◽  
Protospacer Adjacent Motif

Background: CRISPR-Cas systems have expanded the possibilities for gene editing in bacteria and eukaryotes. There are many excellent tools for designing the CRISPR-Cas guide RNAs for model organisms with standard Cas enzymes. GuideMaker is intended as a fast and easy-to-use design tool for atypical projects with 1) non-standard Cas enzymes, 2) non-model organisms, or 3) projects that need to design a panel of guide RNAs (gRNA) for genome-wide screens. Findings: GuideMaker can rapidly design gRNAs for gene targets across the genome from a degenerate protospacer adjacent motif (PAM) and a GenBank file. The tool applies Hierarchical Navigable Small World (HNSW) graphs to speed up the comparison of guide RNAs. This allows the user to design gRNAs targeting all genes in a typical bacterial genome in about 1-2 minutes. Conclusions: Guidemaker 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 smaller 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.

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

2015 ◽  
Author(s):  
Nathan Wong ◽  
Weijun Liu ◽  
Xiaowei Wang
Keyword(s):  
Genome Editing ◽  
Web Server ◽  
Design Tool ◽  
Rna Seq ◽  
Bioinformatics Tool ◽  
Guide Rna ◽  
Guide Rnas ◽  
Bioinformatics Tools ◽  
Genome Wide

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).

scholarly journals Improving the Precision of Base Editing by Bubble Hairpin Single Guide RNA

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Zhiwei Hu ◽  
Yannan Wang ◽  
Qian Liu ◽  
Yan Qiu ◽  
Zhiyu Zhong ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Breaks ◽  
Single Nucleotide ◽  
Base Editing ◽  
Guide Rna ◽  
Guide Rnas ◽  
Double Stranded Dna ◽  
Target Sites ◽  
Guide Sequence ◽  
Sgrna Design

ABSTRACT Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5′ end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency. Meanwhile, such a design also improves the purity of C-to-T conversions induced by base editor 3 (BE3) at on-target sites. Our results present a distinctive and effective strategy to improve the specificity of base editing. IMPORTANCE Base editors are DSB-free genome editing tools and have been widely used in diverse living systems. However, it is reported that these tools can cause substantial off-target editings. To meet this challenge, we developed a new approach to improve the specificity of base editors by using hairpin sgRNAs with a bubble. Furthermore, our sgRNA design also dramatically reduced indels and unwanted base substitutions at on-target sites. We believe that the BH-sgRNA design is a significant improvement over existing sgRNAs of base editors, and our design promises to be adaptable to various base editors. We expect that it will make contributions to improving the safety of gene therapy.

scholarly journals Crisflash: open-source software to generate CRISPR guide RNAs against genomes annotated with individual variation

2019 ◽  
Vol 35 (17) ◽  
pp. 3146-3147 ◽  
Author(s):  
Adrien L S Jacquin ◽  
Duncan T Odom ◽  
Margus Lukk
Keyword(s):  
Open Source Software ◽  
Software Tool ◽  
Supplementary Information ◽  
Small Scale ◽  
Genome Sequences ◽  
Guide Rnas ◽  
Genome Modification ◽  
Order Of Magnitude ◽  
Sgrna Design ◽  
Reference Genomes

Abstract Summary CRISPR/Cas9 system requires short guide RNAs (sgRNAs) to direct genome modification. Most currently available tools for sgRNA design operate only with standard reference genomes, and are best suited for small-scale projects. To address these limitations, we developed Crisflash, a software tool for fast sgRNA design and potential off-target discovery, built for performance and flexibility. Crisflash can rapidly design CRISPR guides against any sequenced genome or genome sequences, and can optimize guide accuracy by incorporating user-supplied variant data. Crisflash is over an order of magnitude faster than comparable tools, even using a single CPU core, and efficiently and robustly scores the potential off-targeting of all possible candidate CRISPR guide oligonucleotides. Availability and implementation https://github.com/crisflash Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals kRISP-meR: A Reference-free Guide-RNA Design Tool for CRISPR/Cas9

2019 ◽  
Author(s):  
Mahmudur Rahman Hera ◽  
Amatur Rahman ◽  
Atif Rahman
Keyword(s):  
Genome Editing ◽  
Design Tool ◽  
Target Region ◽  
Guide Rna ◽  
Guide Rnas ◽  
Rna Design ◽  
Reference Genomes ◽  
Target Effects

AbstractGenome editing using the CRISPR/Cas9 system requires designing guide RNAs (sgRNA) that are efficient and specific. Guide RNAs are usually designed using reference genomes which limits their use in organisms with no or incomplete reference genomes. Here, we present kRISP-meR, a reference free method to design sgRNAs for CRISPR/Cas9 system. kRISP-meR takes as input a target region and sequenced reads from the organism to be edited and generates sgRNAs that are likely to minimize off-target effects. Our analysis indicates that kRISP-meR is able to identify majority of the guides identified by a widely used sgRNA designing tool, without any knowledge of the reference, while retaining specificity.

scholarly journals CRISPR as a Potential Technique to Reduce Suicide Risk

2021 ◽  
Vol 10 (3) ◽  
Author(s):  
Hidemi Zamora ◽  
Javier Cornejo
Keyword(s):  
Cystic Fibrosis ◽  
Suicide Risk ◽  
Gene Editing ◽  
Design Tool ◽  
High Importance ◽  
Knock Out ◽  
Guide Rna ◽  
Guide Rnas ◽  
The Past ◽  
Rna Design

As suicide is the nineteenth leading cause of death worldwide, it is important to focus on discovering ways to reduce the risk of suicide-related death as much as possible. With CRISPR starting to become increasingly popular over the past few years, this gene editing technique has been used to study how to edit, turn off, or knock out multiple parts of the genome. However, research on genes related to diseases as cystic fibrosis or Alzheimer’s disease has been mainly prioritized and, even though they are of high importance as well, important issues such as suicide have been left into oblivion. Four genes have been proven to be key in influencing suicide risk, showing that not only environmental factors account for an increased possibility of death by this cause. Therefore, gene editing techniques such as CRISPR could be applied in order to knock out those genes and reduce this risk. This research used Synthego’s guide RNA design tool to predict how the use of CRISPR can be helpful in knocking out those four suicide-related genes and, consequently, in preventing suicide. The top-ranked guide RNAs for each gene were used, showing the best results possible and with the least number of off-targets, which, in turn, demonstrates the effectiveness of CRISPR as a potential technique to reduce the number of suicide-related deaths worldwide.

scholarly journals beditor: A computational workflow for designing libraries of guide RNAs for CRISPR-mediated base editing

2018 ◽  
Author(s):  
Rohan Dandage ◽  
Philippe C Després ◽  
Nozomu Yachie ◽  
Christian R Landry
Keyword(s):  
A Priori ◽  
Maximum Activity ◽  
Base Editing ◽  
Guide Rna ◽  
Guide Rnas ◽  
Genome Wide ◽  
Disease Mutations ◽  
Computational Workflow ◽  
Activity Window ◽  
Target Effects

ABSTRACTCRISPR-mediated base editors have opened unique avenues for scar-free genome-wide mutagenesis. Here, we describe a comprehensive computational workflow called beditor that can be broadly adapted for designing guide RNA libraries with a range of CRISPR-mediated base editors, PAM recognition sequences and genomes of many species. Additionally, in order to assist users in selecting the best sets of guide RNAs for their experiments, a priori estimates, called beditor scores are calculated. These beditor scores are intended to select guide RNAs that conform to requirements for optimal base editing: the editable base falls within maximum activity window of the CRISPR-mediated base editor and produces non-confounding mutational effects with minimal predicted off-target effects. We demonstrate the utility of the software by designing guide RNAs for base-editing to create or remove thousands of clinically important human disease mutations.

scholarly journals PrimeDesign software for rapid and simplified design of prime editing guide RNAs

2020 ◽  
Author(s):  
Jonathan Y. Hsu ◽  
Andrew V. Anzalone ◽  
Julian Grünewald ◽  
Kin Chung Lam ◽  
Max W. Shen ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Web Application ◽  
Saturation Mutagenesis ◽  
Command Line ◽  
Searchable Database ◽  
Guide Rna ◽  
Guide Rnas ◽  
Genome Wide ◽  
Command Line Tool ◽  
User Friendly

AbstractPrime editing (PE) is a versatile genome editing technology, but design of the required guide RNAs is more complex than for standard CRISPR-based nucleases or base editors. Here we describe PrimeDesign, a user-friendly, end-to-end web application and command-line tool for the design of PE experiments. PrimeDesign can be used for single and combination editing applications, as well as genome-wide and saturation mutagenesis screens. Using PrimeDesign, we also constructed PrimeVar, the first comprehensive and searchable database for prime editing guide RNA (pegRNA) and nicking sgRNA (ngRNA) combinations to install or correct >68,500 pathogenic human genetic variants from the ClinVar database.

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