Project description:A key goal of developmental biology is to understand how a single cell transforms into a full-grown organism consisting of many cells. Although impressive progress has been made in lineage tracing using imaging approaches, analysis of vertebrate lineage trees has mostly been limited to relatively small subsets of cells. Here we present scar-trace, a strategy for massively parallel whole-organism lineage tracing based on Cas9 induced genetic scars in the zebrafish.
Project description:The pairing of CRISPR/Cas9-based gene editing with massively parallel single-cell readouts now enables large-scale lineage tracing. However, the rapid growth in complexity of data from these assays has outpaced our ability to accurately infer phylogenetic relationships. First, we introduce Cassiopeia - a suite of scalable maximum parsimony approaches for tree reconstruction. Second, we provide a simulation framework for evaluating algorithms and exploring lineage tracer design principles. Finally, we generate the most complex experimental lineage tracing dataset to date, 34,557 human cells continuously traced over 15 generations, and use it for benchmarking phylogenetic inference approaches. We show that Cassiopeia outperforms traditional methods by several metrics and under a wide variety of parameter regimes, and provide insight into the principles for the design of improved Cas9-enabled recorders. Together these should broadly enable large-scale mammalian lineage tracing efforts.Cassiopeia and its benchmarking resources are publicly available at https://www.github.com/YosefLab/Cassiopeia.
Project description:To identify direct transcriptional targets of RFX6, we performed chromatin immunoprecipitation of HA epitope tagged RFX6 followed by massively parallel DNA sequencing (ChIP-seq). Using CRISPR/Cas9 gene editing, the HA epitope was inserted into the 3' end of the RFX6 gene in H9 hESC. Pluripotent cells were then differentiated into PDX1+RFX6+ pancreatic progenitors and endogenous RFX6-HA was immunoprecipitated with an anti-HA antibody. To eliminate background signal caused by non-specific antibody binding, a control experiment using wild-type H9 hESC was performed in parallel.
