Guide RNAs with embedded barcodes boost CRISPR-pooled screens.

Area of Science:

• Molecular Biology
• Genetics
• Bioinformatics

Background:

• CRISPR screening is a powerful tool for gene function analysis.
• Conventional CRISPR screens can suffer from high false-positive and false-negative rates.
• High multiplicity of infection (MOI) can exacerbate screening inefficiencies.

Purpose of the Study:

• To introduce a novel method for enhancing CRISPR screening accuracy and efficiency.
• To address limitations of conventional CRISPR screens, particularly at high MOI.
• To provide a robust system for cell-limited or in vivo screening applications.

Main Methods:

• Development of re-designed guide RNAs incorporating internal barcodes (iBARs) within their loop regions.
• Comparative analysis of iBAR approach versus conventional CRISPR screening methods.
• Evaluation of performance across varying multiplicities of infection (MOI).

Main Results:

• The iBAR approach significantly reduces false-positive and false-negative rates compared to conventional methods.
• iBARs enable highly efficient and accurate screening at high MOI with reduced cell input.
• Demonstrated superior performance over canonical CRISPR screens at low MOI.

Conclusions:

• The iBAR system offers a substantial improvement in CRISPR screening reliability and efficiency.
• This method is particularly advantageous for experiments with limited cell availability or challenging in vivo settings.
• iBARs represent a significant advancement for genetic screening technologies.