NIS-Seq enables cell-type-agnostic optical perturbation screening.

Area of Science:

• Cell biology
• Genomics
• High-throughput screening

Background:

• Optical pooled screening uses microscopy to link cell phenotypes with perturbations.
• Existing methods are limited by cell type, transcriptional activity, and density.
• Cytosolic detection of barcoded transcripts restricts application.

Purpose of the Study:

• To introduce and validate Nuclear In-Situ Sequencing (NIS-Seq) for broad-scale optical screening.
• To overcome limitations of previous optical screening methods.
• To identify key players in inflammation-related cellular pathways.

Main Methods:

• Developed NIS-Seq by integrating an inverted phage promoter for sgRNA amplification in the nucleus.
• Sequenced barcoded genomic DNA directly from nuclei, independent of cellular transcription.
• Benchmarked NIS-Seq across eight cell types and four genome-scale screens.
• Performed pooled optical screens in primary human macrophages and skin tissue.

Main Results:

• NIS-Seq successfully screened nucleated cells at high density and library complexity.
• Demonstrated robust barcode identification in diverse cell types, including primary human tissues.
• Identified key regulators in inflammation-related cellular pathways through genome-scale screens.

Conclusions:

• NIS-Seq is a versatile and scalable technology for optical pooled screening.
• The method expands the applicability of optical screening to various cell types and tissues.
• NIS-Seq facilitates discovery of novel cellular pathway components.