Online High-throughput Mutagenesis Designer Using Scoring Matrix of Sequence-specific Endonucleases.

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• CRISPR-Cas9 and other sequence-specific endonucleases are crucial for genome editing, gene knockout, and gene therapy.
• A need exists for efficient computational tools to support high-throughput gene mutagenesis pipelines.

Purpose of the Study:

• To develop an automated system, the Cas9 Online Designer (COD), for screening Cas9 targets and off-targets.
• To provide gene knockout and genotyping strategies for genome editing applications.

Main Methods:

• The COD system was developed to screen Cas9 targets and off-targets.
• It generates gene knockout and genotyping strategies.
• An off-target scoring matrix applicable to various genome editing tools was incorporated.

Main Results:

• A gene knockout rat model was successfully created and genotyped, validating COD's predictive accuracy for targets and off-targets.
• The system designed gene knockout strategies for 72 rat cytochrome P450 genes instantly, demonstrating high-throughput efficiency.
• The COD system proved capable of supporting sequence-specific endonuclease-induced mutagenesis.

Conclusions:

• The COD system provides a speedy, accurate, and flexible computational pipeline for gene knockout.
• It effectively supports high-throughput gene mutagenesis using sequence-specific endonucleases.
• The system's off-target scoring matrix has broader applicability beyond Cas9.