Advanced single-cell pooled CRISPR screening identifies C19orf53 required for cell proliferation based on mTORC1...

Area of Science:

• Genomics
• Molecular Biology
• Cell Biology

Background:

• Multiplexed single-cell CRISPR screening accelerates biological discovery.
• CRISPR gene knockout efficiency has limitations, including off-target effects.

Purpose of the Study:

• Introduce DoNick-seq, an advanced method to improve gene knockout and reduce off-target activity in CRISPR screening.
• Enable simultaneous genomic perturbation and transcriptome profiling at the single-cell level.
• Investigate mTORC1 regulators and their role in cell growth and amino acid metabolism.

Main Methods:

• Re-engineered plasmid constructs for pooled CRISPR screening of single-cell transcriptomes.
• Applied DoNick-seq to probe mTORC1 regulators in two cell lines.
• Analyzed genomic perturbation and transcriptome profiles from over 20,000 single cells.

Main Results:

• DoNick-seq efficiently identified gene targets, cell numbers, and cellular profiles.
• Characterized mTORC1 negative and positive regulators, revealing insights into cell growth and inhibition.
• Demonstrated mTORC1 hyperactivation exhausts free amino acids via increased proliferation.
• Identified C19orf53 as a gene mediating excessive proliferation, metabolic imbalance, and enhanced oxidative stress.

Conclusions:

• DoNick-seq facilitates high-throughput functional dissection of complex cellular responses at the single-cell level.
• Increases the accuracy of CRISPR single-cell transcriptomics.
• Provides new understanding of mTORC1 signaling, cell growth, and metabolic regulation.