scholarly journals Ares-GT: design of guide RNAs targeting multiple genes for CRISPR-Cas experiments

2020 ◽  
Author(s):  
Eugenio G. Minguet
Keyword(s):  
Reference Genome ◽  
Query Sequence ◽  
Gene Families ◽  
Guide Rna ◽  
Guide Rnas ◽  
Online Tools ◽  
Multiple Input ◽  
Design Guide ◽  
Command Line Tool ◽  
Selection Of

ABSTRACTMotivationThere is a lack of tools to design guide RNA for CRISPR genome editing of gene families and usually good candidate sgRNAs are tagged with low scores precisely because they match several locations in the genome, thus time-consuming manual evaluation of targets is required. Moreover, online tools are limited to a restricted list of reference genome and lack the flexibility to incorporate unpublished genomes or contemplate genomes of populations with allelic variants.ResultsTo address these issues, I have developed the ARES-GT, a local command line tool in Python software. ARES-GT allows the selection of candidate sgRNAs that match multiple input query sequences, in addition of candidate sgRNAs that specifically match each query sequence. It also contemplates the use of unmapped contigs apart from complete genomes thus allowing the use of any genome provided by user and being able to handle intraspecies allelic variability and individual polymorphisms.AvailabilityARES-GT is available at GitHub (https://github.com/eugomin/ARES-GT.git).

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.

scholarly journals GRIBCG: A software for selection of sgRNAs in the design of balancer chromosomes

2018 ◽  
Author(s):  
Brian B. Merritt ◽  
Lily S. Cheung
Keyword(s):  
Rational Design ◽  
Chromosomal Rearrangements ◽  
Fruit Fly ◽  
Model Organisms ◽  
Guide Rna ◽  
Guide Rnas ◽  
Genetics Research ◽  
Multicellular Organisms ◽  
Balancer Chromosomes ◽  
Selection Of

AbstractBackgroundBalancer chromosomes are tools used by fruit fly geneticists to prevent meiotic recombination. Recently, CRISPR/Cas9 genome editing has been shown capable of generating inversions similar to the chromosomal rearrangements present in balancer chromosomes. Extending the benefits of balancer chromosomes to other multicellular organisms could significantly accelerate biomedical and plant genetics research.ResultsHere, we present GRIBCG (Guide RNA Identifier for Balancer Chromosome Generation), a tool for the rational design of balancer chromosomes. GRIBCG identifies single guide RNAs (sgRNAs) for use with Streptococcus pyogenes Cas9 (SpCas9). These sgRNAs would efficiently cut a chromosome multiple times while minimizing off-target cutting in the rest of the genome. We describe the performance of this tool on six model organisms and compare our results to two routinely used fruit fly balancer chromosomes.ConclusionGRIBCG is the first of its kind tool for the design of balancer chromosomes using CRISPR/Cas9. GRIBCG can accelerate genetics research by providing a fast, systematic and simple to use framework to induce chromosomal rearrangements.

scholarly journals Faster and better CRISPR guide RNA design with the Crackling method

2020 ◽  
Author(s):  
Jacob Bradford ◽  
Timothy Chappell ◽  
Dimitri Perrin
Keyword(s):  
Experimental Data ◽  
Task Design ◽  
Design Tools ◽  
Guide Rna ◽  
Guide Rnas ◽  
Design Guide ◽  
Order Of Magnitude ◽  
Software Distribution ◽  
Rna Design ◽  
Better Than

The design of CRISPR-Cas9 guide RNAs is not trivial, and is a computationally demanding task. Design tools need to identify target sequences that will maximise the likelihood of obtaining the desired cut, whilst minimising off-target risk. There is a need for a tool that can meet both objectives while remaining practical to use on large genomes.Here, we present Crackling, a new method that is more suitable for meeting these objectives. We test its performance on 12 genomes and on data from validation studies. Crackling maximises guide efficiency by combining multiple scoring approaches. On experimental data, the guides it selects are better than those selected by others. It also incorporates Inverted Signature Slice Lists (ISSL) for faster off-target scoring. ISSL provides a gain of an order of magnitude in speed, while preserving the same level of accuracy. Overall, this makes Crackling a faster and better method to design guide RNAs at scale.Crackling is available at https://github.com/bmds-lab/Crackling under the Berkeley Software Distribution (BSD) 3-Clause license.

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

2020 ◽  
Author(s):  
Jonathan Hsu ◽  
Julian Grünewald ◽  
Regan Szalay ◽  
Justine Shih ◽  
Andrew Anzalone ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Web Application ◽  
Saturation Mutagenesis ◽  
Command Line ◽  
Guide Rna ◽  
Guide Rnas ◽  
Pathogenic Variants ◽  
Genome Wide ◽  
Command Line Tool ◽  
User Friendly

Abstract Prime 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 constructed PrimeVar, a comprehensive and searchable database that includes candidate prime editing guide RNA (pegRNA) and nicking sgRNA (ngRNA) combinations for installing or correcting >68,500 pathogenic human genetic variants from the ClinVar database. Finally, we used PrimeDesign to design pegRNAs/ngRNAs to install a variety of human pathogenic variants in human cells.

scholarly journals Nucleolar Factors Direct the 2′-O-Ribose Methylation and Pseudouridylation of U6 Spliceosomal RNA

1999 ◽  
Vol 19 (10) ◽  
pp. 6906-6917 ◽  
Author(s):  
Philippe Ganot ◽  
Beáta E. Jády ◽  
Marie-Line Bortolin ◽  
Xavier Darzacq ◽  
Tamás Kiss
Keyword(s):  
Small Nuclear Rna ◽  
Guide Rna ◽  
Guide Rnas ◽  
Trafficking Pathway ◽  
5.8S Rrna ◽  
Nuclear Rna ◽  
Nucleolar Rna ◽  
Correct Target ◽  
Selection Of ◽  
U6 Rna

ABSTRACT The nucleolus has long been known as a functionally highly specialized subnuclear compartment where synthesis, posttranscriptional modification, and processing of cytoplasmic rRNAs take place. In this study, we demonstrate that the nucleolus contains all thetrans-acting factors that are responsible for the accurate and efficient synthesis of the eight 2′-O-methylated nucleotides and three pseudouridine residues carried by the mammalian U6 spliceosomal small nuclear RNA. Factors mediating the formation of pseudouridine residues in the U3 small nucleolar RNA are also present and functionally active in the nucleolus. For selection of the correct target nucleotides in the U6 and U3 RNAs, the nucleolar 2′-O-methylation and pseudouridylation factors rely on short sequences located around the target nucleotide to be modified. This observation further underscores a recently proposed role for small nucleolar guide RNAs in the 2′-O-methylation of the U6 spliceosomal RNA (K. T. Tycowski, Z.-H. You, P. J. Graham, and J. A. Steitz, Mol. Cell 2:629–638, 1998). We demonstrate that a novel 2′-O-methylated nucleotide can be generated in the yeast U6 RNA by use of an artificial 2′-O-methylation small nucleolar guide RNA. We also show that a short fragment of the 5.8S rRNA, when expressed as part of the human U6 RNA, is faithfully 2′-O-methylated and pseudouridylated. These results are most consistent with a trafficking pathway in which the U6 spliceosomal RNA cycles through the nucleolus to undergo nucleolar RNA-directed modifications.

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

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jonathan Y. Hsu ◽  
Julian Grünewald ◽  
Regan Szalay ◽  
Justine Shih ◽  
Andrew V. Anzalone ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Web Application ◽  
Saturation Mutagenesis ◽  
Command Line ◽  
Guide Rna ◽  
Guide Rnas ◽  
Pathogenic Variants ◽  
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 construct PrimeVar, a comprehensive and searchable database that includes candidate prime editing guide RNA (pegRNA) and nicking sgRNA (ngRNA) combinations for installing or correcting >68,500 pathogenic human genetic variants from the ClinVar database. Finally, we use PrimeDesign to design pegRNAs/ngRNAs to install a variety of human pathogenic variants in human cells.

ASSESSMENT OF EFFICIENCY AND OFF-TARGET ACTIVITY OF CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES

2020 ◽  
Author(s):  
Y.V. Mikhaylova ◽  
◽  
M.A. Tyumentseva ◽  
A.A. Shelenkov ◽  
Y.G. Yanushevich ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Correct Choice ◽  
Target Sequence ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Chemokine Receptor Ccr5 ◽  
Target Activity

In this study, we assessed the efficiency and off-target activity of the CRISPR/CAS complex with one of the selected guide RNAs using the CIRCLE-seq technology. The gene encoding the human chemokine receptor CCR5 was used as a target sequence for genome editing. The results of this experiment indicate the correct choice of the guide RNA and efficient work of the CRISPR- CAS ribonucleoprotein complex used. CIRCLE-seq technology has shown high sensitivity compared to bioinformatic methods for predicting off-target activity of CRISPR/CAS complexes. We plan to evaluate the efficiency and off-target activity of CRISPR/CAS ribonucleoprotein complexes with other guide RNAs by slightly adjusting the CIRCLE-seq-technology protocol in order to reduce nonspecific DNA breaks and increase the number of reliable reads.

scholarly journals How to Use Online Tools to Generate New Hypotheses for Mammary Gland Biology Research: A Case Study for Wnt7b

2021 ◽  
Author(s):  
Yorick Bernardus Cornelis van de Grift ◽  
Nika Heijmans ◽  
Renée van Amerongen
Keyword(s):  
Mammary Gland ◽  
In Silico ◽  
Online Tools ◽  
Mammary Gland Biology ◽  
User Friendly ◽  
In Silico Analyses ◽  
Biology Research ◽  
Selection Of

AbstractAn increasing number of ‘-omics’ datasets, generated by labs all across the world, are becoming available. They contain a wealth of data that are largely unexplored. Not every scientist, however, will have access to the required resources and expertise to analyze such data from scratch. Fortunately, a growing number of investigators is dedicating their time and effort to the development of user friendly, online applications that allow researchers to use and investigate these datasets. Here, we will illustrate the usefulness of such an approach. Using regulation of Wnt7b expression as an example, we will highlight a selection of accessible tools and resources that are available to researchers in the area of mammary gland biology. We show how they can be used for in silico analyses of gene regulatory mechanisms, resulting in new hypotheses and providing leads for experimental follow up. We also call out to the mammary gland community to join forces in a coordinated effort to generate and share additional tissue-specific ‘-omics’ datasets and thereby expand the in silico toolbox.

scholarly journals Haplotype-resolved genome of diploid ginger (Zingiber officinale) and its unique gingerol biosynthetic pathway

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Hong-Lei Li ◽  
Lin Wu ◽  
Zhaoming Dong ◽  
Yusong Jiang ◽  
Sanjie Jiang ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Southwest China ◽  
Reference Genome ◽  
Zingiber Officinale ◽  
Gene Families ◽  
Chromosome Conformation ◽  
Long Reads ◽  
Transcription Factor Networks ◽  
Species Specific ◽  
Haplotype 1

AbstractGinger (Zingiber officinale), the type species of Zingiberaceae, is one of the most widespread medicinal plants and spices. Here, we report a high-quality, chromosome-scale reference genome of ginger ‘Zhugen’, a traditionally cultivated ginger in Southwest China used as a fresh vegetable, assembled from PacBio long reads, Illumina short reads, and high-throughput chromosome conformation capture (Hi-C) reads. The ginger genome was phased into two haplotypes, haplotype 1 (1.53 Gb with a contig N50 of 4.68 M) and haplotype 0 (1.51 Gb with a contig N50 of 5.28 M). Homologous ginger chromosomes maintained excellent gene pair collinearity. In 17,226 pairs of allelic genes, 11.9% exhibited differential expression between alleles. Based on the results of ginger genome sequencing, transcriptome analysis, and metabolomic analysis, we proposed a backbone biosynthetic pathway of gingerol analogs, which consists of 12 enzymatic gene families, PAL, C4H, 4CL, CST, C3’H, C3OMT, CCOMT, CSE, PKS, AOR, DHN, and DHT. These analyses also identified the likely transcription factor networks that regulate the synthesis of gingerol analogs. Overall, this study serves as an excellent resource for further research on ginger biology and breeding, lays a foundation for a better understanding of ginger evolution, and presents an intact biosynthetic pathway for species-specific gingerol biosynthesis.

scholarly journals An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

2021 ◽  
Author(s):  
Eugene V. Gasanov ◽  
Justyna Jędrychowska ◽  
Michal Pastor ◽  
Malgorzata Wiweger ◽  
Axel Methner ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Target Site ◽  
Proof Of Concept ◽  
Intervention Site ◽  
End Joining ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Non Homologous End Joining

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

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