Developmental barcoding of whole mouse via homing CRISPR

Abstract

In vivo barcoding using nuclease-induced mutations is a powerful approach for recording biological information, including developmental lineages; however, its application in mammalian systems has been limited. We present in vivo barcoding in the mouse with multiple homing guide RNAs that each generate hundreds of mutant alleles and combine to produce an exponential diversity of barcodes. Activation upon conception and continued mutagenesis through gestation resulted in developmentally barcoded mice wherein information is recorded in lineage-specific mutations. We used these recordings for reliable post hoc reconstruction of the earliest lineages and investigation of axis development in the brain. Our results provide an enabling and versatile platform for in vivo barcoding and lineage tracing in a mammalian model system.

An engineered CRISPR/Cas9 mouse line for simultaneous readout of lineage histories and gene expression profiles in single cells

“…Finally, this work among others (Alemany et al, 2018;Chan et al, 2019;Frieda et al, 2017;Kalhor et al, 2018;Raj et al, 2018;Spanjaard et al, 2018) represents a proof-of-principle study for the robust recording of cellular information using genome editing. In principle, CARLIN can be extended so that Cas9 expression is controlled by environmentally-sensitive promoters rather than doxycycline.…”

“…Innovations that have increased our ability to both modify and detect DNA sequences in single cells has led to sophisticated lineage tracing systems based on CRISPR-Cas9 gene editing, with recent implementations in mice (Chan et al, 2019;Kalhor et al, 2018). In these previously described models, constitutive Cas9 expression generates evolvable barcodes in hundreds of random genomic target sites.…”

“…Modified variants of this system that use expressed barcodes have allowed for the simultaneous measurement of singlecell gene expression levels and lineage tracing (Alemany et al, 2018;Raj et al, 2018;Spanjaard et al, 2018). More recently, systems combining delivery of expressed barcodes with transposons in the mouse embryo have been described (Chan et al, 2019;Kalhor et al, 2018). Both of these approaches use constitutively expressed Cas9 and use multiple target arrays (barcodes) to generate diversity.…”

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

“…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.…”

Inference of Single-Cell Phylogenies from Lineage Tracing Data

“…Another potentially promising design consideration concerns the range of character muta- 23 tion rates and their variability across different target sites -a parameter that can be precisely 24 engineered [30]. In this design, one would expect the variability to help distinguish between early 25 and late branching points and consequently achieve better resolution of the underlying phylogeny 26 [51,31,32]. We simulated "Phased Recorders" ( Figure S22) with varying levels of target-site cut- 27 ting variability and observe that this design allows for better inference when the distributions of 28 mutation probabilities are more dispersed ( Figure S22b).…”