An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Summary

Here, we present a method for generating tissue-specific binary transcription systems in Drosophila by replacing the first coding exon of genes with transcription drivers. The CRISPR/Cas9-based method places a transactivator sequence under the endogenous regulation of a replaced gene, and consequently facilitates transctivator expression exclusively in gene-specific spatiotemporal patterns.

Abstract

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of cells or tissues in model organisms. These systems contain two components as separate transgenic lines. A driver line expresses a transcriptional activator under the control of tissue-specific promoters/enhancers, and a reporter/effector line harbors a target gene placed downstream to the binding site of the transcription activator. Animals harboring both components induce tissue-specific transactivation of a target gene expression. Precise spatiotemporal expression of the gene in targeted tissues is critical for unbiased interpretation of cell/gene activity. Therefore, developing a method for generating exclusive cell/tissue-specific driver lines is essential. Here we present a method to generate highly tissue-specific targeted expression system by employing a "Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated" (CRISPR/Cas)-based genome editing technique. In this method, the endonuclease Cas9 is targeted by two chimeric guide RNAs (gRNA) to specific sites in the first coding exon of a gene in the Drosophila genome to create double-strand breaks (DSB). Subsequently, using an exogenous donor plasmid containing the transactivator sequence, the cell-autonomous repair machinery enables homology-directed repair (HDR) of the DSB, resulting in precise deletion and replacement of the exon with the transactivator sequence. The knocked-in transactivator is expressed exclusively in cells where the cis-regulatory elements of the replaced gene are functional. The detailed step-by-step protocol presented here for generating a binary transcriptional driver expressed in Drosophila fgf/branchless-producing epithelial/neuronal cells can be adopted for any gene- or tissue-specific expression.

Introduction

The genetic toolbox for targeted gene expression has been well developed in Drosophila, making it one of the best model systems to investigate the function of genes involved in a wide variety of cellular processes. Binary expression systems, such as yeast Gal4/UAS (upstream activation sequence), was first adopted for tissue-specific enhancer trapping and gene misexpression in the Drosophila genetic model¹ (Figure 1). This system facilitated the development of a large number of techniques such as spatiotemporal regulation of gene overexpression, misexpression, knockout in selected groups o....

Protocol

1. Designing and Constructing the gRNA Expression Vector

1. To precisely replace a long defined region of an exon, use a dual gRNA approach⁶, in which each gRNA can specifically target two ends of the selected region of interest. To obtain an accurate gene-specific spatiotemporal expression of the driver, select two gRNA target sites within the first coding exon of the gene.
2. For Drosophila melanogaster, select the gRNA target sites using the flyCRISPR Optimal Target Finder tool (http://tools.flycrispr.molbio.wisc.edu/targetFinder/). Other available CRISPR design tools can be used as well, including the Optimi....

Results

This protocol was successfully used to generate a targeted binary expression reporter system specific for bnl expressing cells⁵. The cis-regulatory elements (CREs) that control complex spatiotemporal bnl expression are not characterized. Therefore, to achieve spatiotemporal expression under the control of the endogenous bnl regulatory sequence, only the first coding exon of bnl was designed to be replaced with the sequen.......

Discussion

Traditionally, Drosophila enhancer traps were generated by two different methods. One of the ways includes random insertion of a driver (eg., Gal4) sequence in the genome by transposition (e.g., P-element transposition)¹ . Alternatively, the driver sequences can be placed under the transcriptional control of a putative enhancer/promoter region in a plasmid construct, which would then be integrated into an ectopic site of the genome^3,

Materials

Name	Company	Catalog Number	Comments
X-Gal/IPTG	Gentrox (Genesee Scientific)	18-218	cloning
LB-Agar	BD Difco	BD 244520	cloning
Tris-HCl	Sigma Aldrich	T3253	Molecular Biology
EDTA	Sigma Aldrich	E1161	Molecular Biology
NaCl	Sigma Aldrich	S7653	Molecular Biology
UltraPure DNase/RNase-Free Water	ThermoFisher Scientific	10977-023	Molecular Biology
10%SDS	Sigma Aldrich	71736	Molecular Biology
KOAc	Fisher-Scientific	P1190	Molecular Biology
EtOH	Fisher-Scientific	04-355-451	Molecular Biology
GeneJET Miniprep	ThermoFisher Scientific	K0503	Miniprep
PureLink HiPure Plasmid Maxipep kits	ThermoFisher Scientific	K210006	Maxiprep
BbsI	NEB	R0539S	Restriction enzyme
Primers	IDT-DNA		PCR
pCFD4	Kornberg Lab		DNA template and vector for gRNA
KAPA HiFi Hot Start- (Kapa Biosystems)	Kapa biosystems	KK2601	PCR
Q5-high fidelity Taq	NEB	NEB #M0491	PCR
Gibson Assembly Master Mix	NEB	NEB #E2611	DNA assembly
pBPnlsLexA:p65Uw	Addgene		DNA template for LexA amplification
Proteinase K	ThermoFisher Scientific	25530049	Molecular Biology
2x PCR PreMix, with dye (red)	Sydlab	MB067-EQ2R	Molecular Biology
Gel elution kit	Zymo Research (Genesee Scientific)	11-300	Molecular Biology
TRI reagent	Sigma-Aldrich		Molecular Biology
Direct-zol RNA purification kits	Zymo Research (Genesee Scientific)	11-330	Molecular Biology
OneTaq One-Step RT-PCR Kit	NEB	E5315S	Molecular Biology
lexO-CherryCAAX	Kornberg Lab		Fly line
UAS-CD8:GFP	Kornberg lab		Fly line
btl-Gal4	Kornberg lab		Fly line
MKRS/TB6B	Kornberg lab		Fly line
Confocal Microscope SP5X	Leica		Imaging expression pattern
CO2 station	Genesee Scientific	59-122WCU	fly pushing
Stereo microscope	Olympus	SZ-61	fly pushing
Microtube homogenizing pestles	Fisher-Scientific	03-421-217	genomic DNA isolation
NanoDrop spectrophotometer	ThermoFisher Scientific	ND-1000	DNA quantification