Quantitative fate mapping: A general framework for analyzing progenitor state dynamics via retrospective lineage barcoding.

Natural and induced somatic mutations that accumulate in the genome during development record the phylogenetic relationships of cells; whether these lineage barcodes capture the complex dynamics of progenitor states remains unclear. We introduce quantitative fate mapping, an approach to reconstruct the hierarchy, commitment times, population sizes, and commitment biases of intermediate progenitor states during development based on a time-scaled phylogeny of their descendants. To reconstruct time-scaled phylogenies from lineage barcodes, we introduce Phylotime, a scalable maximum likelihood clustering approach based on a general barcoding mutagenesis model. We validate these approaches using realistic in silico and in vitro barcoding experiments. We further establish criteria for the number of cells that must be analyzed for robust quantitative fate mapping and a progenitor state coverage statistic to assess the robustness. This work demonstrates how lineage barcodes, natural or synthetic, enable analyzing progenitor fate and dynamics long after embryonic development in any organism.

Post-translation digital data encoding into the genomes of mammalian cell populations.

High resolution cellular signal encoding is critical for better understanding of complex biological phenomena. DNA-based biosignal encoders alter genomic or plasmid DNA in a signal dependent manner. Current approaches involve the signal of interest affecting a DNA edit by interacting with a signal specific promoter which then results in expression of the effector molecule (DNA altering enzyme). Here, we present the proof of concept of a biosignal encoding system where the enzyme terminal deoxynucleotidyl transferase (TdT) acts as the effector molecule upon directly interacting with the signal of interest. A template independent DNA polymerase (DNAp), TdT incorporates nucleotides at the 3' OH ends of DNA substrate in a signal dependent manner. By employing CRISPR-Cas9 to create double stranded breaks in genomic DNA, we make 3'OH ends available to act as substrate for TdT. We show that this system can successfully resolve and encode different concentrations of various biosignals into the genomic DNA of HEK-293T cells. Finally, we develop a simple encoding scheme associated with the tested biosignals and encode the message "HELLO WORLD" into the genomic DNA of HEK-293T cells at a population level with 91% accuracy. This work demonstrates a simple and engineerable system that can reliably store local biosignal information into the genomes of mammalian cell populations.

Lineage barcoding in mice with homing CRISPR.

Classic approaches to mapping the developmental history of cells in vivo have relied on techniques that require complex interventions and often capture only a single trajectory or moment in time. We have previously described a developmental barcoding system to address these issues using synthetically induced mutations to record information about each cell's lineage in its genome. This system uses MARC1 mouse lines, which have multiple homing guide RNAs that each generate hundreds of mutant alleles and combine to produce an exponential diversity of barcodes. Here, we detail two MARC1 lines that are available from a public repository. We describe strategies for using MARC1 mice and experimental design considerations. We provide a protocol for barcode retrieval and sequencing as well as the analysis of the sequencing data. This protocol generates barcodes based on synthetically induced mutations in mice to enable lineage analysis.

A comprehensive library of human transcription factors for cell fate engineering.

Human pluripotent stem cells (hPSCs) offer an unprecedented opportunity to model diverse cell types and tissues. To enable systematic exploration of the programming landscape mediated by transcription factors (TFs), we present the Human TFome, a comprehensive library containing 1,564 TF genes and 1,732 TF splice isoforms. By screening the library in three hPSC lines, we discovered 290 TFs, including 241 that were previously unreported, that induce differentiation in 4 days without alteration of external soluble or biomechanical cues. We used four of the hits to program hPSCs into neurons, fibroblasts, oligodendrocytes and vascular endothelial-like cells that have molecular and functional similarity to primary cells. Our cell-autonomous approach enabled parallel programming of hPSCs into multiple cell types simultaneously. We also demonstrated orthogonal programming by including oligodendrocyte-inducible hPSCs with unmodified hPSCs to generate cerebral organoids, which expedited in situ myelination. Large-scale combinatorial screening of the Human TFome will complement other strategies for cell engineering based on developmental biology and computational systems biology.

Developmental barcoding of whole mouse via homing CRISPR.

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.