An Engineered CRISPR-Cas9 Mouse Line for Simultaneous Readout of Lineage Histories and Gene Expression Profiles in Single Cells

Abstract

Tracing the lineage history of cells is key to answering diverse and fundamental questions in biology. Particularly in the context of stem cell biology, analysis of single cell lineages in their native state has elucidated novel fates and highlighted heterogeneity of function. Coupling of such ancestry information with other molecular readouts represents an important goal in the field. Here, we describe the CARLIN (for CRISPR Array Repair LINeage tracing) mouse line and corresponding analysis tools that can be used to simultaneously interrogate the lineage and transcriptomic information of single cells in vivo. This model exploits CRISPR technology to generate up to 44,000 transcribed barcodes in an inducible fashion at any point during development or adulthood, is compatible with sequential barcoding, and is fully genetically defined. We have used CARLIN to identify intrinsic biases in the activity of fetal liver hematopoietic stem cell (HSC) clones and to uncover a previously unappreciated clonal bottleneck in the response of HSCs to injury. CARLIN also allows the unbiased identification of transcriptional signatures based on in vivo stem cell function without a need for markers or cell sorting..

Single-cell lineage and transcriptome reconstruction of metastatic cancer reveals selection of aggressive hybrid EMT states

“…3a). Deletions predominated over insertions as expected 4,6,9 , with an approximately equal number of single-and multi-target deletions (Fig. 3b, Extended Data Fig.…”

“…Overall, 89% of transcriptomes had corresponding clonal lineage information for M1 and 77% for M2, demonstrating improved barcode recovery using macsGESTALT compared to prior methods 6,9 . In total, across all sites in both mice, we recovered both the transcriptome and clonal history for 28,028 single cells (M1: 12,657; M2: 15,371) (Extended Data Fig.…”

“…Such methods can be readily coupled to single-cell RNA sequencing (scRNA-seq) to explicitly relate cell lineage histories with transcriptional states [6][7][8] . Until recently 9,10 , GESTALT and related methods have primarily been applied to early development, e.g. by injection of CRISPR/Cas9 reagents into zygotes and subsequent profiling of edited barcodes and single cell transcriptomes from the resulting multicellular organism.…”

Single-cell genomics to understand disease pathogenesis

“…Recently, on the basis of the concept that daughter cells generally have the same genome, lineage tracing analysis using DNA barcode technology, a method of lineage identification that uses a short section of re-writable DNA, has been advancing [37]. Approaches for generating DNA barcodes include retrovirus-induced genome insertion [38,39], Cre/loxPmediated recombination [40][41][42], and CRISPR/Cas9-mediated DNA double-strand breaks [43][44][45][46]. Several approaches can read out barcode information as mRNA molecules, enabling the simultaneous detection of gene expression and lineage information [42,45,46].…”

Dense reconstruction of elongated cell lineages: overcoming suboptimum lineage encoding and sparse cell sampling

“…To separate peaks for cell types with broad, potentially mixed distributions, it may be reasonable to resolve the cell production window(s) by increasing the number of coding units. Alternatively, one can target discrete temporal windows for stage-specific lineage tracing via temporal induction of Cas9 [15,44]. This partial lineage tracing should separate otherwise indistinguishable cells based on the timing of their birth.…”