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Development of a graphical user interface for sgRNAcas9 and its application
Chang-zhi Zhao1, Yi Zhang2, Guang-lei Li1
1Key Lab of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China.
Yi Chuan = Hereditas
|October 27, 2015
Summary
Designing single guide RNA (sgRNA) with CRISPR/Cas9 is crucial for minimizing off-target effects. The sgRNAcas9 software, now with a user-friendly interface, efficiently designs sgRNAs for microRNA precursors across species.
Area of Science:
- Genomics
- Molecular Biology
- Bioinformatics
Background:
- CRISPR/Cas9 is a powerful genome editing tool.
- Off-target effects of Cas9 nuclease activity are a significant concern.
- Designing single guide RNA (sgRNA) with minimal off-target effects is critical for reliable CRISPR applications.
Purpose of the Study:
- To develop a graphical user interface (GUI) for the sgRNAcas9 software package.
- To enhance the usability of sgRNAcas9 for designing sgRNAs and evaluating potential off-target cleavage sites.
- To assess the off-target effects of sgRNAs based on mismatched number and seed sequence specifications.
Main Methods:
- Development of a Java-based GUI for the sgRNAcas9 software.
- Evaluation of sgRNA off-target effects using mismatch number and seed sequence criteria.
- Application of sgRNAcas9 to design sgRNAs targeting 4691 microRNA precursors across multiple species (human, mouse, rat, pig, chicken).
- In vitro validation of on/off-target activity for a sgRNA targeting human miR-206 precursor using T7E1 assay.
Main Results:
- The developed GUI simplifies the usage of the sgRNAcas9 program.
- sgRNAcas9 successfully designed 34,124 sgRNAs for microRNA precursors.
- Off-target effects were evaluated, and sgRNA GC content ranged from 40% to 60%.
- The T7E1 assay confirmed the on/off-target activity of the selected sgRNA.
Conclusions:
- The sgRNAcas9 software, with its new GUI, provides an accessible tool for designing sgRNAs with minimal off-target effects for various species.
- The software is suitable for designing sgRNAs targeting the majority of microRNA precursors.
- The study validates the utility and effectiveness of sgRNAcas9 in CRISPR/Cas9 genome editing applications.
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