Lineage tracing using a Cas9-deaminase barcoding system targeting endogenous L1 elements

Hwang, Byung Kook; Lee, Wookjae; Yum, Soo‐Young; Jeon, Yujin; Cho, Namjin; Jang, Goo; Bang, Duhee

doi:10.1038/s41467-019-09203-z

Cited by 47 publications

(32 citation statements)

References 30 publications

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“…Due to obvious similarity in the problem settings, algorithms from phylogeny such as maximum parsimony [11] and neighbor joining [12] were tried. In addition, custom graph-based algorithm (LINNAEUS [8]), or hierarchical clustering with some specic distance metric and linkage method were tried ( [10,3]). However, whether there are any dierences between these, one of them is the best, or there is other algorithm better than all of these is not clear.…”

Section: Faithful Reconstruction Of Densely Encoded Cell Lineagesmentioning

confidence: 99%

“…For continuous tracking, the earlier DNA edits should not be erased by later edits. Using CRISPR, we can derive genetic barcodes through editing pre-selected sites that may exist as clustered exogeneous gRNA targets [15,8,6] or distribute widely , like transposon-related repeats, in the genome [10]. As to tandem targets, one major concern is interference across nearby targets.…”

Section: Engineering Crispr Barcodes In Vivomentioning

confidence: 99%

“…Targeting endogenous repeats, by contrast, should better ensure independent edits. Encouragingly,one recent study has demonstrated the feasibility of editing endogenous L1 elements in tracking lineages of cultured cells[10]. However, involving endogenous sites requires detailed characterization of the genome to identify ideal targets (in terms of editing eciency and edit retrievability) that play no role in normal development.…”

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confidence: 99%

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Theoretical modeling on CRISPR-coded cell lineages: efficient encoding and optimal reconstruction

Sugino

García-Marqués

Espinosa-Medina

et al. 2019

Preprint

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Delineating cell lineages is a prerequisite for understanding the genesis of cell types. Recent studies have demonstrated the feasibility of generating and reconstructing CRISPR/Cas9-coded cell lineages. However, these works have not investigated the limitations or optimality of the encoding or reconstruction processes.Here, we surveyed a multitude of reconstruction algorithms and found hierarchical clustering, with a metric based on the number of shared Cas9 edits, provides the best reconstruction. As to the eciency, the simple encoding method, with constant Cas9/gRNA edit rate, produces exponential reduction in available coding units and severely limits the trackable depth of lineages. To overcome this, we propose alternative encoding methods, one based on parallel gRNA cascades enabled by CLADES, and another based on variable Cas9 editing rate. Both signicantly increase the trackable depth. In summary, we provide a theoretical basis in understanding, designing and analyzing ecient and robust CRISPR-based cell lineage tracking system.

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Section: Faithful Reconstruction Of Densely Encoded Cell Lineagesmentioning

confidence: 99%

Section: Engineering Crispr Barcodes In Vivomentioning

confidence: 99%

mentioning

confidence: 99%

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Theoretical modeling on CRISPR-coded cell lineages: efficient encoding and optimal reconstruction

Sugino

García-Marqués

Espinosa-Medina

et al. 2019

Preprint

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“…The 10-unit array yielded enormous barcode diversity but suffered from limited duration that it can track due to transient Cas9 activity and rapid coding unit exhaustion. Labs have since worked to increase the encoding capacity by a number of techniques [16,13,17,29]. Studies have also attempted to extend the duration of the encoding process.…”

Section: Introductionmentioning

confidence: 99%

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

Sugino

Miyares

Espinosa-Medina

et al. 2020

Preprint

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Acquiring both lineage and cell-type information during brain development could elucidate transcriptional programs underling neuronal diversification. This is now feasible with single-cell RNA-seq combined with CRISPR-based lineage tracing, which generates genetic barcodes with cumulative CRISPR edits. This technique has not yet been optimized to deliver high-resolution lineage reconstruction of protracted lineages. Drosophila neuronal lineages are an ideal model to consider, as multiple lineages have been morphologically mapped at single-cell resolution. Here we find the parameter ranges required to encode a representative neuronal lineage emanating from 100 stem cell divisions. We derive the optimum editing rate to be inversely proportional to lineage depth, enabling encoding to persist across lineage progression. Further, we experimentally determine the editing rates of a Cas9-deaminase in cycling neural stem cells, finding near ideal rates to map elongated Drosophila neuronal lineages. Moreover, we propose and evaluate strategies to separate recurring cell-types for lineage reconstruction. Finally, we present a simple method to combine multiple experiments, which permits dense reconstruction of protracted cell lineages despite suboptimum lineage encoding and sparse cell sampling.

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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] .…”

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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Lineage tracing using a Cas9-deaminase barcoding system targeting endogenous L1 elements

Cited by 47 publications

References 30 publications

Theoretical modeling on CRISPR-coded cell lineages: efficient encoding and optimal reconstruction

Theoretical modeling on CRISPR-coded cell lineages: efficient encoding and optimal reconstruction

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

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

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