PINCER: improved CRISPR/Cas9 screening by efficient cleavage at conserved residues

Area of Science:

• Genomics
• Molecular Biology
• Drug Discovery

Background:

• CRISPR/Cas9 screens are vital for identifying drug targets.
• Current CRISPR libraries are limited by inefficient gene knockout guides.
• Existing guide design strategies (Cas9 cleavage optimization, domain targeting) are used independently.

Purpose of the Study:

• To develop a cohesive guide design strategy for improved gene knockout.
• To create a novel genome-wide CRISPR library integrating multiple design features.
• To enhance the sensitivity and efficacy of CRISPR screening for drug discovery.

Main Methods:

• Massive reanalysis of CRISPR tiling data using an extensive feature database.
• Development of a guide design algorithm combining enzymatic efficiency and conserved protein region targeting.
• Incorporation of additional features: domains, coding sequence position, termination signals, restriction sites, polymorphisms, and specificity.

Main Results:

• Introduction of the ProteIN ConsERvation (PINCER) genome-wide CRISPR library.
• PINCER uniquely integrates enzymatic efficiency optimization with conserved protein region targeting.
• Head-to-head validation demonstrates superior performance of the PINCER library over existing alternatives.

Conclusions:

• The PINCER library offers a significantly improved strategy for gene knockout.
• This advancement enhances the reliability and sensitivity of functional genomic screens.
• PINCER is available for human and mouse genomes, supporting individual gene knockout and genome-wide screening.