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

Area of Science:

• Molecular Biology
• Genetics
• Neuroscience

Background:

• CRISPR-based gene activation (CRISPRa) offers therapeutic potential for gene therapy by upregulating gene expression.
• Targeting promoters or enhancers in a tissue/cell-type specific manner is crucial for effective gene therapy.

Approach:

• Developed an experimental framework combining highly multiplexed perturbations with single-cell RNA sequencing (sc-RNA-seq).
• Introduced random combinations of guide RNAs (gRNAs) to cells to identify CRISPRa-responsive cis-regulatory elements and their target genes.
• Applied the method to K562 cells and iPSC-derived excitatory neurons using a library of 493 gRNAs.

Key Points:

• Identified gRNAs that specifically upregulate intended target genes without affecting neighboring genes within 1 Mb.
• Discovered gRNAs capable of upregulating six autism spectrum disorder (ASD) and neurodevelopmental disorder (NDD) risk genes in neurons.
• Observed that enhancer responsiveness to CRISPRa is cell-type restricted, dependent on chromatin landscape and trans-acting factors.

Conclusions:

• The described method facilitates large-scale screens for gRNAs that activate therapeutically relevant genes in a cell type-specific manner.
• This approach advances the development of targeted gene therapies for various conditions, including neurodevelopmental disorders.