Multiplex, single-cell CRISPRa screening for cell type specific regulatory elements.

Area of Science:

• Genomics
• Molecular Biology
• Gene Regulation

Background:

• CRISPR-based gene activation (CRISPRa) enables targeted upregulation of gene expression.
• Identifying cell-type-specific regulatory elements is crucial for understanding gene control.
• Existing methods lack the resolution to map precise regulatory element function across diverse cell types.

Purpose of the Study:

• To develop and validate an experimental framework for identifying cell-type-specific CRISPRa-responsive cis-regulatory elements.
• To discover regulatory elements that precisely control the expression of neighboring genes within a defined genomic region.
• To investigate the cell-type specificity of enhancer activity using CRISPRa.

Main Methods:

• A highly multiplexed CRISPRa perturbation system was combined with single-cell RNA sequencing (sc-RNA-seq).
• Random combinations of guide RNAs (gRNAs) targeting candidate cis-regulatory elements were introduced into cells.
• Cells were profiled using sc-RNA-seq, and perturbations were analyzed to assess effects on gene expression in test versus control groups.

Main Results:

• The framework successfully identified gRNAs that specifically upregulate intended target genes without affecting neighboring genes within 1 Mb.
• CRISPRa-responsive enhancers demonstrated cell-type-specific activity, indicating dependence on cellular context.
• Upregulation of six autism spectrum disorder (ASD) and neurodevelopmental disorder (NDD) risk genes in neurons was achieved using this method.

Conclusions:

• The developed framework enables efficient discovery of cell-type-specific regulatory elements and their target genes.
• Enhancer activity is highly dependent on the cell type, influenced by chromatin landscape and trans-acting factors.
• This approach facilitates large-scale screening for CRISPRa tools to activate genes with precise cell-type specificity.