CROPseq-multi: a universal solution for multiplexed perturbation in high-content pooled CRISPR screens.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Forward genetic screens are crucial for understanding biological systems by analyzing gene perturbations and resulting phenotypes.
• Multiplexing genetic perturbations enhances screening efficiency and enables the study of complex genetic interactions.
• Existing multiplexing tools often conflict with pooled screening methods requiring mRNA-embedded barcodes, limiting their use in techniques like single-cell sequencing.

Purpose of the Study:

• To develop a novel lentiviral system, CROPseq-multi, for multiplexing Streptococcus pyogenes (Sp) Cas9-based perturbations with mRNA-embedded barcodes.
• To ensure compatibility with pooled screening methodologies, including those using single-cell sequencing.
• To improve the efficiency and applicability of multiplexed genetic screens.

Main Methods:

• Development of a CROPseq-inspired lentiviral system (CROPseq-multi).
• Integration of Streptococcus pyogenes (Sp) Cas9 for genetic perturbations.
• Incorporation of mRNA-embedded barcodes for pooled screening compatibility.
• Optimization of an in situ detection protocol for optical pooled screens.

Main Results:

• CROPseq-multi demonstrates equivalent per-guide activity to CROPseq with low lentiviral recombination.
• The system is compatible with enrichment screening and optical pooled screens.
• Optimized protocols improved barcode detection efficiency 10-fold and decoding efficiency 3-fold for optical pooled screens.
• CROPseq-multi is extensible to single-cell sequencing readouts.

Conclusions:

• CROPseq-multi provides a versatile solution for multiplexed genetic screening using SpCas9.
• This advancement broadens the applicability of pooled screening methodologies, particularly those requiring mRNA-embedded barcodes.
• Facilitates deeper investigation into genetic interactions and complex biological systems.