Multiplex profiling of developmental cis-regulatory elements with quantitative single-cell expression reporters.
Nat Methods
; 21(6): 983-993, 2024 Jun.
Article
in En
| MEDLINE
| ID: mdl-38724692
ABSTRACT
The inability to scalably and precisely measure the activity of developmental cis-regulatory elements (CREs) in multicellular systems is a bottleneck in genomics. Here we develop a dual RNA cassette that decouples the detection and quantification tasks inherent to multiplex single-cell reporter assays. The resulting measurement of reporter expression is accurate over multiple orders of magnitude, with a precision approaching the limit set by Poisson counting noise. Together with RNA barcode stabilization via circularization, these scalable single-cell quantitative expression reporters provide high-contrast readouts, analogous to classic in situ assays but entirely from sequencing. Screening >200 regions of accessible chromatin in a multicellular in vitro model of early mammalian development, we identify 13 (8 previously uncharacterized) autonomous and cell-type-specific developmental CREs. We further demonstrate that chimeric CRE pairs generate cognate two-cell-type activity profiles and assess gain- and loss-of-function multicellular expression phenotypes from CRE variants with perturbed transcription factor binding sites. Single-cell quantitative expression reporters can be applied in developmental and multicellular systems to quantitatively characterize native, perturbed and synthetic CREs at scale, with high sensitivity and at single-cell resolution.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Gene Expression Regulation, Developmental
/
Single-Cell Analysis
Limits:
Animals
/
Humans
Language:
En
Journal:
Nat Methods
Year:
2024
Document type:
Article