A mouse model with high clonal barcode diversity for joint lineage, transcriptomic, and epigenomic profiling in single cells

Cellular lineage histories along with their molecular states encode fundamental principles of tissue development and homeostasis. Current lineage-recording mouse models have limited barcode diversity and poor single-cell lineage coverage, thus precluding their use in tissues composed of millions of cells. Here, we developed DARLIN, an improved Cas9 barcoding mouse line that utilizes terminal deoxynucleotidyl transferase (TdT) to enhance insertion events over 30 CRISPR target sites, stably integrated into 3 distinct genomic loci. DARLIN is inducible, has an estimated ~10^18 lineage barcodes across tissues, and enables detection of usable barcodes in ~60% of profiled single cells. Using DARLIN, we examined fate priming within developing hematopoietic stem cells (HSCs) and revealed unique features of HSC migration. Additionally, we adapted a method to jointly profile DNA methylation, chromatin accessibility, gene expression, and lineage information in single cells. Using this approach we found that cellular clonal memory is associated with genome-wide DNA methylation rather than gene expression or chromatin accessibility. DARLIN will enable widespread high-resolution study of lineage relationships and their molecular signatures in diverse tissues and physiological contexts. In this study, we generate bulk CARLIN sequence datasets for both the DARLIN and Cas9/CARLIN mouse lines to evaluate the performance of DARLIN. Then, we generated both single-cell CARLIN and transcriptome data to study early fate bias within hematopoietic stem cells (HSCs). Furthermore, we generated bulk CARLIN sequences across different developmental stages to study HSC migration. We also performed single-cell multi-omic lineage tracing (DNA methylation, chromatin accessibility, gene expression, and lineage information) to study the clonal memory of HSCs. The processed data from 10X single-cell RNA sequencing of hematopoietic cells sourced from various bones is accessible through GSE221501. For the single-cell Camellia-seq datasets spanning Hematopoietic Stem Cells (HSCs) across the AGM, fetal liver, and adult bone marrow stages, the processed scRNA-seq data can be accessed via GSE222246; additionally, the processed data for chromatin accessibility (CpG file) and DNA methylation (GpC) can be found at GSE222328. However, the processed versions of the DARLIN lineage datasets linked with these investigations have not been provided. All original sequencing data, including the DARLIN lineage data, associated with our research have been deposited on the SRA server.