PALINCODE: Recording cell lineage with ternary palindromic CRISPR bits

Reconstructing complete and accurate lineage trees remains a long-standing challenge in biology. Here, we introduce PALINCODE (Palindromic Coding and Decoding), a system that utilizes ternary CRISPR bits (cBits) to stochastically write one of three possible states over time, permanently embedding lineage relationships in the genome. We demonstrate PALINCODE's lineage-recording potential through simulations and cell culture models. We show that truncated Cas9 guide sequences yield ternary outcomes at high efficiency when compared to conventional guides. Using PALINCODE, we derived lineage-recording cell lines with a theoretical coding capacity of up to 1025 bits, enabling the generation of lineage trees 32 cell divisions deep in single-cell sequencing of 293T cells. Furthermore, we applied PALINCODE using an in vivo melanoma model to jointly read out lineage history and gene expression, enabling in vivo reconstruction of clonal evolution within tumor cell clonal populations. PALINCODE circumvents several limitations of prior CRISPR-based systems and achieves a higher lineage-recording density than many competing approaches.