BASELINE: a CRISPR base editing platform for mammalian-scale single-cell lineage tracing

Area of Science:

• Molecular Biology
• Genetics
• Developmental Biology

Background:

• Cellular fate is determined by inherited lineage and environmental context.
• CRISPR-based lineage recording technologies offer potential but face challenges in single-cell recovery, engineering, and scalability.
• Accurate lineage tracing is crucial for understanding development and disease progression.

Purpose of the Study:

• To introduce BASELINE, a novel CRISPR-based system for high-resolution lineage tracing.
• To overcome limitations of existing lineage recording technologies.
• To enable detailed mapping of cellular lineages in complex biological systems.

Main Methods:

• Utilized base editing with the Cas12a adenine base editor for irreversible nucleotide editing.
• Designed target arrays with 50 synthetic target sites integrated multiple times into the genome.
• Combined lineage recording with single-cell profiling for comprehensive analysis.

Main Results:

• BASELINE achieved high-resolution lineage tree generation.
• Recorded over 4300 bits of information in a pancreatic cancer model, a 50-fold increase over existing methods.
• Demonstrated high-fidelity capture of recorders, averaging 29 cell divisions per lineage, suitable for mammalian development.

Conclusions:

• BASELINE offers a significant advancement in lineage tracing technology.
• The system is robust across biologically relevant timescales and provides unprecedented information density.
• Expected to be widely applicable to diverse lineage-tracing studies in development and disease, particularly in challenging, distributed systems.