A CRISPR/Cas9-based enhancement of high-throughput single-cell transcriptomics.

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• Single-cell RNA sequencing (scRNAseq) faces challenges in detecting low-abundance transcripts due to interference from highly abundant ones.
• This limitation obscures cellular heterogeneity and hinders the discovery of subtle biological signatures within cell populations.

Purpose of the Study:

• To introduce single-cell CRISPRclean (scCLEAN), a molecular method designed to enhance transcript detection in scRNAseq.
• To demonstrate scCLEAN's capability to improve the discovery of biologically relevant transcripts, particularly those expressed at low levels.

Main Methods:

• scCLEAN employs CRISPR/Cas9 technology to selectively target and remove highly abundant transcripts from scRNAseq libraries.
• This process recomposes the library, redistributing sequencing reads to focus on less abundant, biologically significant transcripts.
• The method was applied to heterogeneous immune cells and homogenous vascular smooth muscle cells, and integrated with single-cell MAS-Seq.

Main Results:

• scCLEAN successfully enhanced the detection of low-abundance transcripts across different cell types and biological contexts.
• The method demonstrated increased transcript-level detection and discovery when applied to third-generation sequencing (single-cell MAS-Seq).
• Experimental applications revealed distinct biological signatures that were previously obscured by abundant transcripts.

Conclusions:

• scCLEAN offers a significant advancement in scRNAseq by overcoming the limitations of transcript abundance variability.
• The technology shows broad utility across various human tissues and cell types, improving the resolution of single-cell transcriptomic analysis.
• The study provides guidance on the optimal contexts for applying scCLEAN to maximize its benefits in biological discovery.