Screening for Genomic Modifications: A Method to Identify CRISPR-Generated Mutants in Drosophila

Abstract

Source: Du, L., et al. An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing. J. Vis. Exp. (2018).

This video describes a screening method to identify transgenic Drosophila flies, whose genome was modified using CRISPR-Cas9. CRISPR-Cas9 is a powerful genome-editing tool that revolutionized the way scientists can manipulate an organism's genome. In the example protocol, we will see how to identify CRISPR-generated mutants, in which an insertion of a transactivation sequence replaces the first exon of the branchless (bnl) gene, thus creating a bnl gene-specific driver line, bnl-LexA.

Protocol

This protocol is an excerpt from Du et al., An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing, J. Vis. Exp. (2018).

1. Fly genetics and screening (Figure 1 and Figure 2)

1. When the injected embryos develop into adults, cross each single G₀ flies to balancer flies. Select suitable balancer for the chromosome containing the targeted allele.
2. Anesthetize the F1 offspring from each G0 cross on a CO₂ pad and randomly pick 10-20 males under a stereomicroscope. Cross them individually to the balancer females as shown ....

Results

Figure 1: An overview of workflow for CRISPR/Cas9-mediated genome editing to generate a binary transcription system. The approximate time duration required for each step is indicated. Please c.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
Primers	IDT-DNA		PCR
Proteinase K	ThermoFisher Scientific	25530049	Molecular Biology
2x PCR PreMix, with dye (red)	Sydlab	MB067-EQ2R	Molecular Biology
MKRS/TB6B	Kornberg lab		Fly line
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