Synthetic recording and in situ readout of lineage information in single cells

Abstract

Reconstructing the lineage relationships and dynamic event histories of individual cells within their native spatial context is a long-standing challenge in biology. Many biological processes of interest occur in optically opaque or physically inaccessible contexts, necessitating approaches other than direct imaging. Here we describe a synthetic system that enables cells to record lineage information and event histories in the genome in a format that can be subsequently read out of single cells in situ. This system, termed memory by engineered mutagenesis with optical in situ readout (MEMOIR), is based on a set of barcoded recording elements termed scratchpads. The state of a given scratchpad can be irreversibly altered by CRISPR/Cas9-based targeted mutagenesis, and later read out in single cells through multiplexed single-molecule RNA fluorescence hybridization (smFISH). Using MEMOIR as a proof of principle, we engineered mouse embryonic stem cells to contain multiple scratchpads and other recording components. In these cells, scratchpads were altered in a progressive and stochastic fashion as the cells proliferated. Analysis of the final states of scratchpads in single cells in situ enabled reconstruction of lineage information from cell colonies. Combining analysis of endogenous gene expression with lineage reconstruction in the same cells further allowed inference of the dynamic rates at which embryonic stem cells switch between two gene expression states. Finally, using simulations, we show how parallel MEMOIR systems operating in the same cell could enable recording and readout of dynamic cellular event histories. MEMOIR thus provides a versatile platform for information recording and in situ, single-cell readout across diverse biological systems.

Inheritance of Cell-Cycle Duration in the Presence of Periodic Forcing

“…Powell, one of the pioneers of single-cells measurements, was described as sitting in a 37°C room for many hours watching bacteria divide and recording manually celldivision events [3]. Recent technological advances in microscopy and microfluidics [4,5] have boosted our ability to gather information over tens of thousands of cells and opened the door to quantitative analyses of the process of cell division on lineages [6,7].…”

Synthetic gene circuits and cellular decision-making in human pluripotent stem cells

“…Traditionally, gene knock-outs, fluorescent labeling of endogenous genes or sensing of transcription factors or signaling pathways have been used to study essential genes that maintain the pluripotent state or lead to cell state transitions [63]. Another focus, that has recently received new attention due to the introduction of CRISPR/Cas9-based approaches [64,65], lies in developing genetic lineage tracking systems to track lineage choices in single live cells [66]. Other genetic methods have been developed to overcome challenges in producing hPSC-derived cells, most common being the defined control of key-transcription factors to manipulate hPSC transitions to differentiated cells.…”

The trickiest family tree in biology

“…Michael Elowitz and Long Cai, both at the California Institute of Technology in Pasadena, have developed a lineage tracer that creates fluorescent probes to help them observe the histories of cells as they develop 8 . Their method can track whether certain developmental genes have been turned on in the past for a given lineage.…”