Guide Swap enables genome-scale pooled CRISPR-Cas9 screening in human primary cells

Area of Science:

• Genomics
• Molecular Biology
• Immunology

Background:

• CRISPR-Cas9 screening is vital for gene function studies.
• Existing methods require stable Cas9 expression, limiting use in human primary cells.
• A new approach is needed for primary cell screening.

Purpose of the Study:

• To develop a novel CRISPR-Cas9 screening method for human primary cells.
• To enable genome-wide gene function discovery in challenging cell types.

Main Methods:

• Developed Guide Swap, a method for CRISPR-Cas9 screening in primary cells.
• Utilized lentivirally delivered guide RNAs (gRNAs) with Cas9 complexed to nontargeting gRNA.
• Validated Guide Swap in CD4+ T cell depletion and enrichment screens.
• Applied Guide Swap to study ex vivo hematopoiesis.

Main Results:

• Guide Swap enables efficient CRISPR-Cas9 editing in human primary cells without stable Cas9 expression.
• Successfully performed genome-scale pooled screening in CD4+ T cells.
• Identified regulators of hematopoietic stem and progenitor cell (HSPC) expansion.

Conclusions:

• Guide Swap is a powerful platform for CRISPR-Cas9 screening in human primary cells.
• This method expands the applicability of CRISPR screening to previously inaccessible cell systems.
• Facilitates discovery of gene function in critical biological processes.