CRISPR-STAT: an easy and reliable PCR-based method to evaluate target-specific sgRNA activity.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR/Cas9 is a powerful genome-engineering tool utilizing Cas9 enzyme and single guide RNA (sgRNA).
• Efficient sgRNA design and pre-screening are crucial for successful targeted mutagenesis and minimizing resource waste.
• Current methods for assessing sgRNA efficacy can be time-consuming or costly.

Purpose of the Study:

• To introduce CRISPR Somatic Tissue Activity Test (CRISPR-STAT), an easy, quick, and cost-effective method for evaluating sgRNA on-target efficiency.
• To validate CRISPR-STAT using zebrafish embryos and sgRNAs with known germline transmission efficiencies.
• To demonstrate the assay's capability for multiplex gene targeting.

Main Methods:

• Development of a fluorescent polymerase chain reaction (PCR)-based assay, CRISPR-STAT.
• Validation in zebrafish embryos injected with 28 different sgRNAs.
• Analysis of somatic activity via fluorescent PCR profiles and correlation with germline transmission efficiency.

Main Results:

• A strong positive correlation was observed between fluorescent PCR profiles in injected embryos and germline transmission efficiency.
• CRISPR-STAT demonstrated sensitivity for evaluating multiplex gene targeting.
• The method proved to be quick, cost-effective, and easy to implement with standard laboratory equipment.

Conclusions:

• CRISPR-STAT is a reliable method for pre-screening sgRNA efficacy in genome editing applications.
• This assay facilitates successful mutagenesis and optimizes resource allocation in research.
• The method is adaptable for various model systems and genome targeting nucleases beyond CRISPR/Cas9 in zebrafish.