Overview of guide RNA design tools for CRISPR-Cas9 genome editing technology

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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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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Enhanced guide-RNA design and targeting analysis for precise CRISPR genome editing of single and consortia of industrially relevant and non-model organisms

Bioinformatics ◽

10.1093/bioinformatics/btx564 ◽

2017 ◽

Vol 34 (1) ◽

pp. 16-23 ◽

Cited By ~ 17

Author(s):

Brian J Mendoza ◽

Cong T Trinh

Keyword(s):

Genome Editing ◽

Model Organisms ◽

Guide Rna ◽

Rna Design

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Advances in guide RNA design for editing plant genomes using CRISPR-Cas systems

Genome editing for precision crop breeding - Burleigh Dodds Series in Agricultural Science ◽

10.19103/as.2020.0082.07 ◽

2021 ◽

pp. 147-174

Author(s):

Kaiyuan Chen ◽

◽

Hao Liu ◽

Kabin Xie ◽

Muhammad Tahir ul Qamar ◽

...

Keyword(s):

Genome Editing ◽

Guide Rna ◽

Plant Genomes ◽

Bioinformatic Tools ◽

General Rules ◽

Target Sites ◽

Rna Design

This chapter discusses the general rules for selecting target sites for genome editing using the CRISPR-Cas technology and summarizes the bioinformatic tools that can be used to design sgRNA sequences.

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Dramatic Enhancement of Genome Editing by CRISPR/Cas9 Through Improved Guide RNA Design

Genetics ◽

10.1534/genetics.115.175166 ◽

2015 ◽

Vol 199 (4) ◽

pp. 959-971 ◽

Cited By ~ 145

Author(s):

Behnom Farboud ◽

Barbara J. Meyer

Keyword(s):

Genome Editing ◽

Guide Rna ◽

Rna Design

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

Nature Communications ◽

10.1038/s41467-019-12281-8 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 21

Author(s):

Daqi Wang ◽

Chengdong Zhang ◽

Bei Wang ◽

Bin Li ◽

Qiang Wang ◽

...

Keyword(s):

Design Tool ◽

High Fidelity ◽

Design Tools ◽

Wild Type ◽

Activity Prediction ◽

Guide Rna ◽

Biological Features ◽

A Genome ◽

Rna Design ◽

Genome Scale

Abstract 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/.

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