Imaging Unique DNA Sequences in Individual Cells Using a CRISPR-Cas9-Based, Split Luciferase Biosensor
OPEN ACCESS
Loading...
Author / Producer
Date
2022-03
Publication Type
Journal Article
ETH Bibliography
yes
Citations
Altmetric
OPEN ACCESS
Data
Rights / License
Abstract
An extensive arsenal of biosensing tools has been developed based on the clustered regularly interspaced short palindromic repeat (CRISPR) platform, including those that detect specific DNA sequences both in vitro and in live cells. To date, DNA imaging approaches have traditionally used full fluorescent reporter-based fusion probes. Such “always-on” probes differentiate poorly between bound and unbound probe and are unable to sensitively detect unique copies of a target sequence in individual cells. Herein we describe a DNA biosensor that provides a sensitive readout for such low-copy DNA sequences through proximity-mediated reassembly of two independently optimized fragments of NanoLuc luciferase (NLuc), a small, bright luminescent reporter. Applying this “turn-on” probe in live cells, we demonstrate an application not easily achieved by fluorescent reporter-based probes, detection of individual endogenous genomic loci using standard epifluorescence microscopy. This approach could enable detection of gene edits during ex vivo editing procedures and should be a useful platform for many other live cell DNA biosensing applications.
Permanent link
Publication status
published
External links
Editor
Book title
Journal / series
Volume
4
Pages / Article No.
867390
Publisher
Frontiers Media
Event
Edition / version
Methods
Software
Geographic location
Date collected
Date created
Subject
CRISPR; live cell imaging; split reporter; single gene locus; dCas9
Organisational unit
09635 - Corn, Jacob / Corn, Jacob