PALINCODE: Recording cell lineage with ternary palindromic CRISPR bits.

Area of Science:

• Genetics
• Molecular Biology
• Bioinformatics

Background:

• Accurate reconstruction of cellular lineage trees is a significant challenge in biology.
• Existing CRISPR-based lineage recording systems have limitations in information capacity and efficiency.

Purpose of the Study:

• To introduce PALINCODE, a novel system for permanent lineage recording using CRISPR.
• To demonstrate the potential of PALINCODE for high-density lineage tracing and clonal evolution studies.

Main Methods:

• Utilized palindromic CRISPR editing with truncated guide sequences for ternary state writing (cBits).
• Developed lineage-recording cell lines with high theoretical coding capacity.
• Applied PALINCODE in cell culture and an in vivo melanoma model.

Main Results:

• Achieved efficient ternary outcomes with truncated Cas9 guide sequences.
• Generated lineage trees up to 32 cell divisions deep in 293T cells.
• Enabled joint readout of lineage history and gene expression in vivo, reconstructing melanoma clonal evolution.

Conclusions:

• PALINCODE offers a high-density lineage recording platform surpassing prior CRISPR-based systems.
• The system circumvents limitations of existing methods, increasing information potential at individual CRISPR sites.
• PALINCODE facilitates advanced studies of clonal evolution and cellular lineage dynamics.