Screening for Genomic Modifications: A Method to Identify CRISPR-Generated Mutants in Drosophila
Published: April 30, 2023
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) 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. Anesthetize the F1 offspr…
Representative 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 click here to view a larger version of this figure. <img alt="Figure 2" class="xfigimg" src="/files/ftp_uplo…
Materials
| 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 |
Cite This Article
Screening for Genomic Modifications: A Method to Identify CRISPR-Generated Mutants in Drosophila. J. Vis. Exp. (Pending Publication), e20110, doi: (2023).