Detection of gene cis-regulatory element perturbations in single-cell transcriptomes

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• CRISPR/Cas9 technology allows precise genome editing.
• Understanding cis-regulatory elements is crucial for gene expression control.
• Single-cell RNA sequencing (scRNA-seq) provides high-resolution transcriptomic data.

Purpose of the Study:

• To introduce poly-adenine CRISPR gRNA-based single-cell RNA-sequencing (pAC-Seq) for direct guide RNA (gRNA) observation in scRNA-seq.
• To assess phenotypic consequences of CRISPR/Cas9-mediated alterations in gene cis-regulatory regions.
• To model the power of detecting regulatory genome-induced transcriptomic effects.

Main Methods:

• Development and application of pAC-Seq.
• Utilizing CRISPR/Cas9 to alter gene cis-regulatory regions.
• Analyzing scRNA-seq data to detect cis-regulatory-induced gene expression alterations.

Main Results:

• pAC-Seq enables direct observation of gRNAs within scRNA-seq data.
• The method detects cis-regulatory alterations affecting target gene expression, even with low biallelic loss (~5%).
• Transcript-targeted sequencing can improve detection sensitivity for low biallelic loss events.

Conclusions:

• pAC-Seq is a powerful tool for studying the functional impact of cis-regulatory elements at single-cell resolution.
• The study provides a model to predict the power of detecting regulatory effects based on loss rates and experimental parameters.
• pAC-Seq facilitates a deeper understanding of gene regulation and its phenotypic consequences.