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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE222486 | GEO | 2023/10/18

REPOSITORIES: GEO

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Project description:Following the removal of implanted mammary tumors, nude mice develop multiple-organ metastases at late stage. The metastases may originate from the primary tumors before the resection surgery, or alternatively, from some established metastases. By multiple approaches, we have proved that bone environment could invigorate cancer cells for further dissemination. this study aims to examine if metastatic dissemination from bone to other sites occurs in natural setting of metastatic spread. We herein apply the rapidly evolving barcode system using homing guide RNA/Cas9 to trace the metastases formation in mouse. hgRNA/Cas9 is a self-targeting Crispr system which allows the mutation occurs in the DNA sequence of guide RNA. Tumor cells wer labelled with doxycycline inducible evolving barcoding system. Upon doxycycline treatment the DNA sequence of hgRNA accumulate mutations with time. The diversity of barcodes in each lesion can infer the timeing of seeding while the mutation patterns of barcodes suggest the phylogenetic correlation of metastases. Several findings were made on this study. First, at the terminal stage, multi-organ metastases are not genetically grouped according to sites of metastases. Nonnegative Matrix Factorization (NMF) analysis of mutant barcodes suggested the early disseminated metastases, which have highest level of Shannon entropy, were featured with a common cluster of mutant barcodes irrespective of their locations. Second, most metastases are potentially multiclonal as indicated by multiple clusters of independent mutant barcodes. Third, when we use Shannon entropy as an index of metastasis age , putative parent-child relationship between metastases with unique mutant barcodes clearly exemplified secondary metastatic seeding from bone to other organs. Finally, we did not observe a clear correlation between tumor burden and Shannon entropy across different metastases, suggesting that putative parental metastases might remain small after seeding further metastases.

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

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