SNP-CRISPR: A Web Tool for SNP-Specific Genome Editing

CRISPR-Cas9 is a powerful genome editing technology in which a single guide RNA (sgRNA) confers target site specificity to achieve Cas9-mediated genome editing. Numerous sgRNA design tools have been developed based on reference genomes for humans and model organisms. However, existing resources are not optimal as genetic mutations or single nucleotide polymorphisms (SNPs) within the targeting region affect the efficiency of CRISPR-based approaches by interfering with guide-target complementarity. To facilitate identification of sgRNAs (1) in non-reference genomes, (2) across varying genetic backgrounds, or (3) for specific targeting of SNP-containing alleles, for example, disease relevant mutations, we developed a web tool, SNP-CRISPR (https://www.flyrnai.org/tools/snp_crispr/). SNP-CRISPR can be used to design sgRNAs based on public variant data sets or user-identified variants. In addition, the tool computes efficiency and specificity scores for sgRNA designs targeting both the variant and the reference. Moreover, SNP-CRISPR provides the option to upload multiple SNPs and target single or multiple nearby base changes simultaneously with a single sgRNA design. Given these capabilities, SNP-CRISPR has a wide range of potential research applications in model systems and for design of sgRNAs for disease-associated variant correction.

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SNP-CRISPR: a web tool for SNP-specific genome editing

10.1101/847277 ◽

2019 ◽

Cited By ~ 1

Author(s):

Chiao-Lin Chen ◽

Jonathan Rodiger ◽

Verena Chung ◽

Raghuvir Viswanatha ◽

Stephanie E. Mohr ◽

...

Keyword(s):

Genome Editing ◽

Model Systems ◽

Model Organisms ◽

Data Sets ◽

Nucleotide Polymorphisms ◽

Web Tool ◽

Guide Rna ◽

Wide Range ◽

Sgrna Design ◽

Reference Genomes

ABSTRACTCRISPR-Cas9 is a powerful genome editing technology in which a single guide RNA (sgRNA) confers target site specificity to achieve Cas9-mediated genome editing. Numerous sgRNA design tools have been developed based on reference genomes for humans and model organisms. However, existing resources are not optimal as genetic mutations or single nucleotide polymorphisms (SNPs) within the targeting region affect the efficiency of CRISPR-based approaches by interfering with guide-target complementarity. To facilitate identification of sgRNAs (1) in non-reference genomes, (2) across varying genetic backgrounds, or (3) for specific targeting of SNP-containing alleles, for example, disease relevant mutations, we developed a web tool, SNP-CRISPR (https://www.flyrnai.org/tools/snp_crispr/). SNP-CRISPR can be used to design sgRNAs based on public variant data sets or user-identified variants. In addition, the tool computes efficiency and specificity scores for sgRNA designs targeting both the variant and the reference. Moreover, SNP-CRISPR provides the option to upload multiple SNPs and target single or multiple nearby base changes simultaneously with a single sgRNA design. Given these capabilities, SNP-CRISPR has a wide range of potential research applications in model systems and for design of sgRNAs for disease-associated variant correction.

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mtDNAcombine: tools to combine sequences from multiple studies

BMC Bioinformatics ◽

10.1186/s12859-021-04048-0 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Eleanor F. Miller ◽

Andrea Manica

Keyword(s):

Sequence Data ◽

Data Extraction ◽

Bayesian Skyline Plot ◽

Model Organisms ◽

Data Sets ◽

Data Handling ◽

Online Database ◽

Genetic Studies ◽

Wide Range ◽

Existing Data

Abstract Background Today an unprecedented amount of genetic sequence data is stored in publicly available repositories. For decades now, mitochondrial DNA (mtDNA) has been the workhorse of genetic studies, and as a result, there is a large volume of mtDNA data available in these repositories for a wide range of species. Indeed, whilst whole genome sequencing is an exciting prospect for the future, for most non-model organisms’ classical markers such as mtDNA remain widely used. By compiling existing data from multiple original studies, it is possible to build powerful new datasets capable of exploring many questions in ecology, evolution and conservation biology. One key question that these data can help inform is what happened in a species’ demographic past. However, compiling data in this manner is not trivial, there are many complexities associated with data extraction, data quality and data handling. Results Here we present the mtDNAcombine package, a collection of tools developed to manage some of the major decisions associated with handling multi-study sequence data with a particular focus on preparing sequence data for Bayesian skyline plot demographic reconstructions. Conclusions There is now more genetic information available than ever before and large meta-data sets offer great opportunities to explore new and exciting avenues of research. However, compiling multi-study datasets still remains a technically challenging prospect. The mtDNAcombine package provides a pipeline to streamline the process of downloading, curating, and analysing sequence data, guiding the process of compiling data sets from the online database GenBank.

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

mBio ◽

10.1128/mbio.00342-21 ◽

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.

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Human genetic variation alters CRISPR-Cas9 on- and off-targeting specificity at therapeutically implicated loci

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1714640114 ◽

2017 ◽

Vol 114 (52) ◽

pp. E11257-E11266 ◽

Cited By ~ 47

Author(s):

Samuel Lessard ◽

Laurent Francioli ◽

Jessica Alfoldi ◽

Jean-Claude Tardif ◽

Patrick T. Ellinor ◽

...

Keyword(s):

Genetic Variation ◽

Treatment Failure ◽

Genome Editing ◽

Wide Spectrum ◽

Adverse Outcomes ◽

Human Genetic Variation ◽

Nucleotide Polymorphisms ◽

Guide Rna ◽

Cas9 Nuclease ◽

Target Sites

The CRISPR-Cas9 nuclease system holds enormous potential for therapeutic genome editing of a wide spectrum of diseases. Large efforts have been made to further understanding of on- and off-target activity to assist the design of CRISPR-based therapies with optimized efficacy and safety. However, current efforts have largely focused on the reference genome or the genome of cell lines to evaluate guide RNA (gRNA) efficiency, safety, and toxicity. Here, we examine the effect of human genetic variation on both on- and off-target specificity. Specifically, we utilize 7,444 whole-genome sequences to examine the effect of variants on the targeting specificity of ∼3,000 gRNAs across 30 therapeutically implicated loci. We demonstrate that human genetic variation can alter the off-target landscape genome-wide including creating and destroying protospacer adjacent motifs (PAMs). Furthermore, single-nucleotide polymorphisms (SNPs) and insertions/deletions (indels) can result in altered on-target sites and novel potent off-target sites, which can predispose patients to treatment failure and adverse effects, respectively; however, these events are rare. Taken together, these data highlight the importance of considering individual genomes for therapeutic genome-editing applications for the design and evaluation of CRISPR-based therapies to minimize risk of treatment failure and/or adverse outcomes.

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AsCRISPR: a web server for allele-specific sgRNA design in precision medicine

10.1101/672634 ◽

2019 ◽

Author(s):

Guihu Zhao ◽

Jinchen Li ◽

Yu Tang

Keyword(s):

Nucleotide Polymorphisms ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Guide Rna ◽

Inherited Diseases ◽

Bioinformatic Tools ◽

Point Of Entry ◽

Allele Specific ◽

Sgrna Design ◽

Specific Restriction

AbstractAllele-specific genomic targeting by CRISPR provides a point of entry for personalized gene therapy of dominantly inherited diseases, by selectively disrupting the mutant alleles or disease-causing single nucleotide polymorphisms (SNPs), ideally while leaving normal alleles intact. Moreover, the allele-specific engineering has been increasingly exploited not only in treating inherited diseases and mutation-driven cancers, but also in other important fields such as genome imprinting, haploinsufficiency, genome loci imaging and immunocompatible manipulations. Despite the tremendous utilities of allele-specific targeting by CRISPR, very few bioinformatic tools have been implemented for the allele-specific purpose. We thus developed AsCRISPR (Allele-specific CRISPR), a web tool to aid the design of guide RNA (gRNA) sequences that can discriminate between alleles. It provides users with limited bioinformatics skills to analyze both their own identified variants and heterozygous SNPs deposited in the dbSNP database. Multiple CRISPR nucleases and their engineered variants including newly-developed Cas12b and CasX are included for users’ choice. Meanwhile, AsCRISPR evaluates the on-target efficiencies, specificities and potential off-targets of gRNA candidates, and also displays the allele-specific restriction enzyme sites that might be disrupted upon successful genome edits. In addition, AsCRISPR analyzed with dominant single nucleotide variants (SNVs) retrieved from ClinVar and OMIM databases, and generated a Dominant Database of candidate discriminating gRNAs that may specifically target the alternative allele for each dominant SNV site. A Validated Database was also established, which manually curated the discriminating gRNAs that were experimentally validated in the mounting literatures. AsCRISPR is freely available at http://www.genemed.tech/ascrispr.

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

10.1101/869115 ◽

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.

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CRISPR-RT: A web service for designing CRISPR-C2c2 crRNA with improved target specificity

10.1101/099895 ◽

2017 ◽

Author(s):

Houxiang Zhu ◽

Emily Richmond ◽

Chun Liang

Keyword(s):

Web Service ◽

Genome Editing ◽

Rna Editing ◽

Model Organisms ◽

Future Research ◽

Target Specificity ◽

Link Type ◽

Wide Range ◽

Set Up

AbstractCRISPR-Cas systems have been successfully applied in genome editing. Recently, the CRISPR-C2c2 system has been reported as a tool for RNA editing. Here we describe CRISPR-RT (CRISPR RNA-Targeting), the first web service to help biologists design the crRNA with improved target specificity for the CRISPR-C2c2 system. CRISPR-RT allows users to set up a wide range of parameters, making it highly flexible for current and future research in CRISPR-based RNA editing. CRISPR-RT covers major model organisms and can be easily extended to cover other species. CRISPR-RT will empower researchers in RNA editing. It is available at http://bioinfolab.miamioh.edu/CRISPR-RT.

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Nucleosomes inhibit target cleavage by CRISPR-Cas9 in vivo

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1810062115 ◽

2018 ◽

Vol 115 (38) ◽

pp. 9351-9358 ◽

Cited By ~ 63

Author(s):

Robert M. Yarrington ◽

Surbhi Verma ◽

Shaina Schwartz ◽

Jonathan K. Trautman ◽

Dana Carroll

Keyword(s):

Genome Editing ◽

Zinc Finger Nucleases ◽

Yeast Genome ◽

Target Sequence ◽

Practical Applications ◽

Guide Rna ◽

Cas9 Protein ◽

Wide Range

Genome editing with CRISPR-Cas nucleases has been applied successfully to a wide range of cells and organisms. There is, however, considerable variation in the efficiency of cleavage and outcomes at different genomic targets, even within the same cell type. Some of this variability is likely due to the inherent quality of the interaction between the guide RNA and the target sequence, but some may also reflect the relative accessibility of the target. We investigated the influence of chromatin structure, particularly the presence or absence of nucleosomes, on cleavage by the Streptococcus pyogenes Cas9 protein. At multiple target sequences in two promoters in the yeast genome, we find that Cas9 cleavage is strongly inhibited when the DNA target is within a nucleosome. This inhibition is relieved when nucleosomes are depleted. Remarkably, the same is not true of zinc-finger nucleases (ZFNs), which cleave equally well at nucleosome-occupied and nucleosome-depleted sites. These results have implications for the choice of specific targets for genome editing, both in research and in clinical and other practical applications.

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Enhanced guide-RNA Design and Targeting Analysis for Precise CRISPR Genome Editing of Single and Consortia of Industrially Relevant and Non-Model Organisms

10.1101/139626 ◽

2017 ◽

Author(s):

Brian J. Mendoza ◽

Cong T. Trinh

Keyword(s):

Metabolic Engineering ◽

Synthetic Biology ◽

Genome Editing ◽

Population Analysis ◽

Strain Engineering ◽

Pathway Engineering ◽

Model Organisms ◽

Guide Rna ◽

Novel Applications ◽

Rna Design

AbstractMotivationGenetic diversity of non-model organisms offers a repertoire of unique phenotypic features for exploration and cultivation for synthetic biology and metabolic engineering applications. To realize this enormous potential, it is critical to have an efficient genome editing tool for rapid strain engineering of these organisms to perform novel programmed functions.ResultsTo accommodate the use of CRISPR/Cas systems for genome editing across organisms, we have developed a novel method, named CASPER (CRISPR Associated Software for Pathway Engineering and Research), for identifying on- and off-targets with enhanced predictability coupled with an analysis of non-unique (repeated) targets to assist in editing any organism with various endonucleases. Utilizing CASPER, we demonstrated a modest 2.4% and significant 30.2% improvement (F-test, p<0.05) over the conventional methods for predicting on- and off-target activities, respectively. Further we used CASPER to develop novel applications in genome editing: multitargeting analysis (i.e. simultaneous multiple-site modification on a target genome with a sole guide-RNA (gRNA) requirement) and multispecies population analysis (i.e. gRNA design for genome editing across a consortium of organisms). Our analysis on a selection of industrially relevant organisms revealed a number of non-unique target sites associated with genes and transposable elements that can be used as potential sites for multitargeting. The analysis also identified shared and unshared targets that enable genome editing of single or multiple genomes in a consortium of interest. We envision CASPER as a useful platform to enhance the precise CRISPR genome editing for metabolic engineering and synthetic biology applications.

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Estimates of introgression as a function of pairwise distances

10.1101/154377 ◽

2017 ◽

Cited By ~ 1

Author(s):

Bastian Pfeifer ◽

Durrell D Kapan

Keyword(s):

R Package ◽

Small Sample ◽

Raw Material ◽

Model Systems ◽

Whole Genome ◽

Nucleotide Polymorphisms ◽

Wide Range ◽

Genomic Scale ◽

Novel Method ◽

Genomic Regions

AbstractBackgroundResearch over the last 10 years highlights the increasing importance of hybridization between species as a major force structuring the evolution of genomes and potentially providing raw material for adaptation by natural and/or sexual selection. Fueled by research in a few model systems where phenotypic hybrids are easily identified, research into hybridization and introgression (the flow of genes between species) has exploded with the advent of whole-genome sequencing and emerging methods to detect the signature of hybridization at the whole-genome or chromosome level. Amongst these are a general class of methods that utilize patterns of single-nucleotide polymorphisms (SNPs) across a tree as markers of hybridization. These methods have been applied to a variety of genomic systems ranging from butterflies to Neanderthal’s to detect introgression, however, when employed at a fine genomic scale these methods do not perform well to quantify introgression in small sample windows.ResultsWe introduce a novel method to detect introgression by combining two widely used statistics: pairwise nucleotide diversity dxy and Patterson’s D. The resulting statistic, the Basic distance fraction (Bdf), accounts for genetic distance across possible topologies and is designed to simultaneously detect and quantify introgression. We also relate our new method to the recently published fd and incorporate these statistics into the powerful genomics R-package PopGenome, freely available on CRAN. The supplemental material contains a wide range of simulation studies and a detailed manual how to perform the statistics within the PopGenome framework.ConclusionWe present a new distance based statistic Bdf that avoids the pitfalls of Patterson’s D when applied to small genomic regions and accurately quantifies the fraction of introgression (f) for a wide range of simulation scenarios.

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