Multiplex genomic recording of enhancer and signal transduction activity in mammalian cells

Abstract

Measurements of gene expression and signal transduction activity are conventionally performed with methods that require either the destruction or live imaging of a biological sample within the timeframe of interest. Here we demonstrate an alternative paradigm, termed ENGRAM (ENhancer-driven Genomic Recording of transcriptional Activity in Multiplex), in which the activity and dynamics of multiple transcriptional reporters are stably recorded to DNA. ENGRAM is based on the prime editing-mediated insertion of signal- or enhancer-specific barcodes to a genomically encoded recording unit. We show how this strategy can be used to concurrently genomically record the relative activity of at least hundreds of enhancers with high fidelity, sensitivity and reproducibility. Leveraging synthetic enhancers that are responsive to specific signal transduction pathways, we further demonstrate time- and concentration-dependent genomic recording of Wnt, NF-κB, and Tet-On activity. Finally, by coupling ENGRAM to sequential genome editing, we show how serially occurring molecular events can potentially be ordered. Looking forward, we envision that multiplex, ENGRAM-based recording of the strength, duration and order of enhancer and signal transduction activities has broad potential for application in functional genomics, developmental biology and neuroscience.

Systematic reconstruction of cellular trajectories across mouse embryogenesis

“…For example, organism-scale lineage recording, originally developed in zebrafish, has recently been adapted to the mouse [91][92][93][94] . Although such methods remain far from delivering the resolution and clarity of the Sulston lineage, they continue to advance from a technical perspective 95,96 . In particular, the concurrent recording of cell lineage and molecular histories may pave the way to more detailed models that explicitly relate patterns of cell division with the unfolding of cell states throughout the developing mouse embryo.…”

Predicting efficiency of writing short sequences into the genome using prime editing

“…Communicated, but as yet unpublished examples include insertion of recombinase sites using prime editing to enable directed insertion of large DNA cargo of up to 36 kb 1,2 , as well as clever utilization of short sequence insertion to generate a molecular recorder for sequential cellular events [40][41][42] . Better understanding of how cellular determinants 17 and pegRNA features affect prime editing rates 14,18 provides a foundation for these advances.…”

A time-resolved, multi-symbol molecular recorder via sequential genome editing

“…However, several groups have engineered gRNAs whose activity is dependent on the binding of specific small molecules or ligands [33][34][35] . Also, we recently developed ENGRAM, a prime editing-based system in which biological signals of interest such as NF-κB and Wnt signals are coupled to the production of specific pegRNAs 36 . These pegRNAs mediate the insertion of signal-specific barcodes to a DNA-based recording site, providing quantitative information with respect to the strength and/or duration of the signal(s).…”

“…c, Schematic of ordered recording via DNA Typewriter. Individual pegRNAs are potentially event driven 36 or constitutively expressed, together with the PE2 enzyme. d-f, Specificity of genome editing on versions of TAPE-1 with two (d), three (e) or five (f) monomers.…”