Precise measurement of molecular phenotypes with barcode-based CRISPRi systems

Area of Science:

• Molecular Biology
• Genetics
• Genomics

Background:

• Genome-wide CRISPR-Cas9 screens are powerful tools for understanding biological processes.
• Effective screens require precise interrogation of molecular phenotypes and discrimination of key regulators.
• Technical factors can distort phenotypic measurements in genetic screens.

Purpose of the Study:

• To develop a generalizable platform, CRISPR interference with barcoded expression reporter sequencing (CiBER-seq), to improve the sensitivity and scope of genome-wide screens.
• To systematically address technical factors affecting phenotypic measurements in CRISPR screens.
• To assess the platform's capability in capturing post-transcriptional and post-translational regulation.

Main Methods:

• Developed CiBER-seq, a CRISPR interference screening platform using barcoded expression reporters.
• Integrated normalized expression reporters with closely-matched control promoters at a single copy.
• Screened for genes affecting nonsense-mediated mRNA decay and Doa10-mediated cytosolic protein decay.

Main Results:

• CiBER-seq significantly enhances the sensitivity and scope of genome-wide screens.
• Normalization against control promoters effectively mitigates technical distortions in phenotypic measurements.
• Optimized CiBER-seq screens accurately identified known components of RNA and protein quality control pathways with low background.

Conclusions:

• CiBER-seq offers a precise and versatile platform for dissecting genetic networks.
• The platform demonstrates high accuracy in capturing post-transcriptional and post-translational regulatory elements.
• CiBER-seq advances the study of cellular behaviors and genetic underpinnings of biological processes.