A multiplexable TALE-based binary expression system for in vivo cellular interaction studies

Abstract

Binary expression systems have revolutionised genetic research by enabling delivery of loss-of-function and gain-of-function transgenes with precise spatial-temporal resolution in vivo. However, at present, each existing platform relies on a defined exogenous transcription activator capable of binding a unique recognition sequence. Consequently, none of these technologies alone can be used to simultaneously target different tissues or cell types in the same organism. Here, we report a modular system based on programmable transcription activator-like effector (TALE) proteins, which enables parallel expression of multiple transgenes in spatially distinct tissues in vivo. Using endogenous enhancers coupled to TALE drivers, we demonstrate multiplexed orthogonal activation of several transgenes carrying cognate variable activating sequences (VAS) in distinct neighbouring cell types of the Drosophila central nervous system. Since the number of combinatorial TALE–VAS pairs is virtually unlimited, this platform provides an experimental framework for highly complex genetic manipulation studies in vivo.

Strategies for Functional Interrogation of Big Cancer Data Using Drosophila Cancer Models

“…Another key advantage Drosophila brings as a functional validation platform for big cancer data is the ability to genetically manipulate multiple genes at once. Some of the tools developed for this purpose include the use of multigenic vectors, multiple short hairpin and guide RNA sequences, multicistronic expression systems that use self-cleaving peptides or Internal Ribosome Entry Sites (IRES), parallel genome editing, and multiplexable orthogonal and intersectional expression systems [16,17,[60][61][62][63][64][65][66][67][68][69]. These tools can be used to build transgenic constructs that capture multiple gene expression changes observed in tumors, or those predicted as candidate biomarkers by computational approaches, and explore their roles in tumorigenesis.…”

Spatial control of gene expression in flies using bacterially derived binary transactivation systems

“…While several binary transactivation systems exist, only a handful have been shown to function in vivo in Drosophila melanogaster (reviewed (Venken et al ., 2011)); therefore, we sought to further expand this powerful molecular genetic tool box. For example, in flies, transactivation systems have been used extensively in vivo affording spatial control including: Gal4‐UAS adapted from yeast (Brand and Perrimon, 1993), the Q‐system adapted from the bread mould Neurospora crassa (Potter et al ., 2010), and several systems derived from bacteria including the LexA/LexAop (Lai and Lee, 2006), the Tet system using tTA/TRE (Bello et al ., 1998), transcription activator‐like effectors (TALEs) (Toegel et al ., 2017), and recently even CRISPR/dCas9‐VPR‐based transactivators (Lin et al ., 2015; Jia et al ., 2018). In addition to spatial control, some of these systems also afford temporal control by exploiting small‐molecule triggers to fine‐tune expression in a dose‐dependent manner.…”

Spatial control of gene expression in flies using bacterially derived binary transactivation systems

“…While several binary transactivation systems exist, only a handful have been shown to function in vivo in Drosophila melanogaster (reviewed (Venken, Simpson, and Bellen 2011)), therefore we sought to further expand this powerful molecular genetic toolbox. For example in flies, transactivation systems have been used extensively in vivo affording spatial control including: Gal4-UAS adapted from yeast (Brand and Perrimon 1993), the Q-system adapted from the bread mold Neurospora crassa (Potter et al 2010), and several systems derived from bacteria including the LexA/LexAop (Lai and Lee 2006), the Tet system using tTA/TRE (Bello, Resendez-Perez, and Gehring 1998), transcription activator-like effectors (TALEs) (Toegel et al 2017), and recently even CRISPR/dCas9-VPR based transactivators (Lin et al 2015;Jia et al 2018). In addition to spatial control, some of these systems also afford temporal control by exploiting smallmolecule triggers to fine-tune expression in a dose-dependent manner.…”