Simultaneous lineage tracing and cell-type identification using CRISPR–Cas9-induced genetic scars

Spanjaard, Bastiaan; Hu, Bo; Mitic, Nina; Olivares-Chauvet, Pedro; Janjuha, Sharan; Ninov, Nikolay; Junker, Jan Philipp

doi:10.1038/nbt.4124

Cited by 458 publications

(436 citation statements)

References 39 publications

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“…However, these methods alone do not track cell dynamics that influence tissue structure, such as timing and location of cell divisions [34-36]. Furthermore, as we show in this study, cell death is common in growth and regeneration, and retrospective cell sequencing excludes cells that do not survive to the point of cell harvest.…”

Section: Discussionmentioning

confidence: 93%

In Toto Imaging of Dynamic Osteoblast Behaviors in Regenerating Skeletal Bone

et al. 2018

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SUMMARY Osteoblasts are matrix-depositing cells that can divide and heal bone injuries. Their deep tissue location and the slow progression of bone regeneration challenge attempts to capture osteoblast behaviors in live tissue at high spatiotemporal resolution. Here, we have developed an imaging platform to monitor and quantify individual and collective behaviors of osteoblasts in adult zebrafish scales, skeletal body armor discs that regenerate rapidly after loss. Using a panel of transgenic lines that visualize and manipulate osteoblasts, we find that a founder pool of osteoblasts emerges through de novo differentiation within one day of scale plucking. These osteoblasts undergo division events that are largely uniform in frequency and orientation to establish a primordium. Osteoblast proliferation dynamics diversify across the primordium by two days after injury, with cell divisions focused near, and with orientations parallel to, the scale periphery, occurring coincident with dynamic localization of fgf20a gene expression. In posterior scale regions, cell elongation events initiate in areas soon occupied by mineralized grooves called radii, beginning approximately 2 days postinjury, with patterned osteoblast death events accompanying maturation of these radii. By imaging at single-cell resolution, we detail acquisition of spatiotemporally distinct cell division, motility, and death dynamics within a founder osteoblast pool as bone regenerates.

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Section: Discussionmentioning

confidence: 93%

In Toto Imaging of Dynamic Osteoblast Behaviors in Regenerating Skeletal Bone

et al. 2018

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“…Independently introduced and evolved mutation patterns in the barcode were analyzed by next‐generation sequencing, by which lineage tracing was achieved. Later, the combination of improved GESTALT (or a related method) and single‐cell RNA‐sequencing (scRNA‐seq) technologies were applied in combinatorial profiling of lineages and cell types . This technology might be appropriate for cancer lineage and stage profiling.…”

Section: A Closer Look At Front‐line Technologiesmentioning

confidence: 99%

“…Later, the combination of improved GESTALT (or a related method) and single-cell RNA-sequencing (scRNA-seq) technologies were applied in combinatorial profiling of lineages and cell types. 54,55 This technology might be appropriate for cancer lineage and stage profiling.…”

Section: Dna Barcoding and Recordingmentioning

confidence: 99%

Acceleration of cancer science with genome editing and related technologies

Sakuma

Yamamoto

2018

Cancer Science

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Genome editing includes various edits of the genome, such as short insertions and deletions, substitutions, and chromosomal rearrangements including inversions, duplications, and translocations. These variations are based on single or multiple DNA double‐strand break (DSB)‐triggered in cellulo repair machineries. In addition to these “conventional” genome editing strategies, tools enabling customized, site‐specific recognition of particular nucleic acid sequences have been coming into wider use; for example, single base editing without DSB introduction, epigenome editing with recruitment of epigenetic modifiers, transcriptome engineering using RNA editing systems, and in vitro detection of specific DNA and RNA sequences. In this review, we provide a quick overview of the current state of genome editing and related technologies that multilaterally contribute to cancer science.

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“…This paradigm of DNA recording has recently seen a transformation with the development of geneticallyencoded CRISPR-based systems that drive rapid mutational accumulation at neutral loci in a cell's genome [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] . When the activity of such systems is linked to the presence of an arbitrary biological stimulus, accumulated mutations become a record of the strength and duration of exposure to the stimulus 3,5,7 ; and when activity is constitutive, accumulated mutations capture lineage relationships among individual cells 2,[4][5][6][11][12][13][14] . Two recent architectures for CRISPR-based recording systems are particularly amenable to recording in extremely large numbers of mammalian cells.…”

Section: Introductionmentioning

confidence: 99%

DNA writing at a single genomic site enables lineage tracing and analog recording in mammalian cells

Tb¹,

Jh²,

Mw³

et al. 2019

Preprint

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The study of intricate cellular and developmental processes in the context of complex multicellular organisms is difficult because it can require the non-destructive observation of thousands, millions, or even billions of cells deep within an animal. To address this difficulty, several groups have recently reported CRISPR-based DNA recorders that convert transient cellular experiences and processes into changes in the genome, which can then be read by sequencing in high-throughput. However, existing DNA recorders act primarily by erasing DNA: they use the random accumulation of CRISPR-induced deletions to record information. This is problematic because in the limit of progressive deletion, no record remains. Here, we present a new type of DNA recorder that acts primarily by writing new DNA. Our system, called CHYRON (Cell HistorY Recording by Ordered iNsertion), inserts random nucleotides at a single locus in temporal order in vivo and can be applied as an evolving lineage tracer as well as a recorder of user-selected cellular stimuli. As a lineage tracer, CHYRON allowed us to perfectly reconstruct the population lineage relationships among 16 groups of human cells descended from four starting groups that were subject to a series of splitting steps. In this experiment, CHYRON progressively wrote and retained base insertions in 20% percent of cells where the average amount written was 8.4 bp (~14.5 bits), reflecting high information content and density. As a stimulus recorder, we showed that when the CHYRON machinery was placed under the control of a stress-responsive promoter, the frequency and length of writing reflected the dose and duration of the stress. We believe CHYRON represents a conceptual advance in DNA recording technologies where writing rather than erasing becomes the primary mode of information accumulation. With further engineering of CHYRON's components to increase writing efficiency, CHYRON should lead to single-cell-resolution recording of lineage and other information through long periods of time in complex animals or tumors, advancing the pursuit of a full picture of mammalian development.

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Simultaneous lineage tracing and cell-type identification using CRISPR–Cas9-induced genetic scars

Cited by 458 publications

References 39 publications

In Toto Imaging of Dynamic Osteoblast Behaviors in Regenerating Skeletal Bone

In Toto Imaging of Dynamic Osteoblast Behaviors in Regenerating Skeletal Bone

Acceleration of cancer science with genome editing and related technologies

DNA writing at a single genomic site enables lineage tracing and analog recording in mammalian cells

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