Mapping the functional impact of non-coding regulatory elements in primary T cells through single-cell CRISPR screens

Area of Science:

• Genomics and Molecular Biology
• Immunology
• Drug Discovery

Background:

• Genetic evidence is crucial for successful drug targeting, yet linking complex disease associations to specific genes remains a challenge.
• Immune disease-associated genetic variants are often found in regulatory elements within T cells, highlighting the need for functional studies.

Purpose of the Study:

• To develop a scalable CRISPR interference (CRISPRi)-based single-cell functional screening approach in primary human T cells.
• To identify genes and molecular programs regulated by non-coding regulatory elements associated with immune diseases.
• To establish a method for mapping regulatory elements to their target genes (E2G) and aid in identifying effector genes for genome-wide association studies (GWAS).

Main Methods:

• Optimized a CRISPRi protocol using CROPseq vectors in primary CD4+ T cells.
• Conducted a large-scale screen targeting 45 regulatory elements and 35 transcription start sites.
• Profiled approximately 250,000 T-cell single-cell transcriptomes and developed a bespoke analytical pipeline for element-to-gene (E2G) mapping.

Main Results:

• The developed pipeline successfully identified both known and novel element-to-gene (E2G) links.
• Integrated genetic association data for immune-related traits, demonstrating the platform's utility.
• Showcased the ability to identify effector genes for GWAS loci, advancing the understanding of disease mechanisms.

Conclusions:

• Introduced 'primary T cell crisprQTL,' a scalable single-cell functional genomics approach for mapping regulatory elements to genes in primary human T cells.
• This framework facilitates the interrogation of immune disease GWAS hits.
• The combination of experimental and quantitative trait loci (QTL)-based techniques is proposed as a solution to the variant-to-function problem in drug discovery.