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 for tissue development and homeostasis. Current cellular barcoding mouse models have limited barcode diversity and poor single-cell lineage readout, 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 allows detection of reliable barcodes in ~60% of profiled single cells. Using DARLIN, we revealed fate priming within hematopoietic stem cells (HSCs) and evaluated HSC migration across tissues. Additionally, we adapted a method to jointly profile DNA methylation, chromatin accessibility, gene expression, and lineage barcodes in single cells. Applying it to study clonal memory of HSCs over time, we found that cells within a clone have more similar genome-wide DNA methylation than gene expression or chromatin accessibility. In total, our study enables systematically dissecting lineage relationships and their molecular mechanisms across diverse problems in biology. Overall design: 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.