acCRISPR: an activity-correction method for improving the accuracy of CRISPR screens

Area of Science:

• Functional genomics
• CRISPR-Cas9 and CRISPR-Cas12a gene editing
• High-throughput screening

Background:

• CRISPR screens are vital for understanding gene function but are challenged by variable guide RNA (sgRNA) cutting efficiency.
• Ineffective sgRNAs can mask the true effects of gene disruption, complicating the identification of essential genes.

Purpose of the Study:

• To develop an end-to-end computational pipeline, acCRISPR, for accurate identification of essential genes in pooled CRISPR screens.
• To correct for sgRNA cutting efficiency variability, thereby improving the reliability of screening outcomes.

Main Methods:

• acCRISPR analyzes sgRNA read counts from next-generation sequencing data.
• It incorporates experimentally determined sgRNA cutting efficiencies to adjust screening results.
• The pipeline calculates an optimization metric to determine gene fitness effects.

Main Results:

• acCRISPR successfully identified essential genes in Yarrowia lipolytica under glucose growth conditions.
• The tool was also applied to identify genes related to salt tolerance in Y. lipolytica.
• A high-confidence set of essential genes was determined for industrial oleochemical production.

Conclusions:

• acCRISPR provides a robust experimental-computational framework for CRISPR-based functional genomics.
• This method enhances the accuracy of essential gene identification in pooled CRISPR screens.
• The framework is adaptable for use in other non-conventional organisms.