Dual-line Genome-scale CRISPR Screening Enables Robust Target Gene Discovery

Area of Science:

• Genomics
• Functional genomics
• Insect molecular biology

Background:

• Pooled CRISPR screening is powerful for genome-scale functional gene discovery.
• Application in insects is limited by systematic differences obscuring functional signals and reliable genetic regulator identification.
• Cas9 genomic integration introduces distinct transcriptional states, editing activity, and screening outcomes, complicating analysis.

Purpose of the Study:

• To develop a robust CRISPR screening framework for insects that overcomes cell line-specific artifacts.
• To improve the reliability and accuracy of functional genomic screening and genetic target discovery in insects.
• To identify novel genetic regulators of insect traits, such as cold tolerance.

Main Methods:

• Developed a dual-line CRISPR screening framework prioritizing genes with concordant effects across independently derived Cas9-expressing insect cell lines.
• Applied the framework to Bactrocera dorsalis for genome-scale functional screening.
• Investigated candidate gene perturbations under cold stress conditions.

Main Results:

• Dual-line consensus screening significantly increased in vivo validation of candidate genes from 60% in single-line screens to 90% in Bactrocera dorsalis.
• Identified candidate regulators of cold tolerance, including a novel gene impacting embryonic survival at low temperatures.
• Demonstrated the framework's ability to filter cell line-specific artifacts.

Conclusions:

• The dual-line CRISPR screening framework enhances the reliability of functional genomic screening in insects.
• This approach facilitates more accurate genetic target discovery for traits like cold tolerance.
• The method provides a powerful tool for dissecting gene function in diverse insect species.