Comparative optimization of combinatorial CRISPR screens

Area of Science:

• Genomics
• Molecular Biology
• Biotechnology

Background:

• Combinatorial CRISPR technologies enable systematic probing of genetic interactions.
• Multiplexing single-guide RNAs (sgRNAs) in CRISPR screens presents performance variability.
• Understanding gene dependencies in redundant gene pairs requires robust functional genomic tools.

Purpose of the Study:

• To benchmark and optimize pooled combinatorial CRISPR libraries for digenic knockout screens.
• To evaluate the performance of different CRISPR-Cas systems and library designs for targeting paralog pairs.
• To identify robust strategies for profiling genetic interactions of multiple genes.

Main Methods:

• Generation and benchmarking of ten distinct pooled combinatorial CRISPR libraries.
• Evaluation of libraries utilizing dual Streptococcus pyogenes Cas9 (spCas9), orthogonal spCas9 and Staphylococcus aureus (saCas9), and enhanced Cas12a.
• Assessment of effect size and positional balance between sgRNAs in digenic knockout screens.

Main Results:

• Significant performance variation observed across different functional genomic tools for multiplexing sgRNAs.
• A combination of alternative tracrRNA sequences with spCas9 demonstrated superior effect size.
• Optimized spCas9 libraries showed improved positional balance between sgRNAs, enhancing reliability.

Conclusions:

• Alternative tracrRNA sequences in spCas9-based combinatorial CRISPR libraries offer a robust approach for genetic interaction profiling.
• Optimized library design is crucial for enhancing the accuracy and efficiency of digenic knockout screens.
• This work provides a foundation for systematic genetic interaction mapping using multiplexed CRISPR technologies.