Editing Transgenic DNA Components by Inducible Gene Replacement in Drosophila melanogaster

Gene conversions occur when genomic double-strand DNA breaks (DSBs) trigger unidirectional transfer of genetic material from a homologous template sequence. Exogenous or mutated sequence can be introduced through this homology-directed repair (HDR). We leveraged gene conversion to develop a method f...

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Bibliographic Details
Published in:Genetics (Austin) 2016-08, Vol.203 (4), p.1613-1628
Main Authors: Lin, Chun-Chieh, Potter, Christopher J
Format: Article
Language:English
Subjects:
Animals
Animals, Genetically Modified - genetics
Chromosomes
Cold
Conversion
CRISPR-Cas Systems - genetics
Deoxyribonucleic acid
DNA
DNA Breaks, Double-Stranded
Drosophila melanogaster
Drosophila melanogaster - genetics
Drosophila Proteins - genetics
Gene Conversion - genetics
Gene Editing
Genetic engineering
Genomes
Hemocytes - metabolism
Insects
Investigations
Methods
Muscles - metabolism
Mutation
Neuroglia - metabolism
Neurons - metabolism
Recombinational DNA Repair - genetics
Transcription factors
Transcription Factors - genetics
Transgenes - genetics
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Summary:Gene conversions occur when genomic double-strand DNA breaks (DSBs) trigger unidirectional transfer of genetic material from a homologous template sequence. Exogenous or mutated sequence can be introduced through this homology-directed repair (HDR). We leveraged gene conversion to develop a method for genomic editing of existing transgenic insertions in Drosophila melanogaster The clustered regularly-interspaced palindromic repeats (CRISPR)/Cas9 system is used in the H: omology A: ssisted C: RISPR K: nock-in (HACK) method to induce DSBs in a GAL4 transgene, which is repaired by a single-genomic transgenic construct containing GAL4 homologous sequences flanking a T2A-QF2 cassette. With two crosses, this technique converts existing GAL4 lines, including enhancer traps, into functional QF2 expressing lines. We used HACK to convert the most commonly-used GAL4 lines (labeling tissues such as neurons, fat, glia, muscle, and hemocytes) to QF2 lines. We also identified regions of the genome that exhibited differential efficiencies of HDR. The HACK technique is robust and readily adaptable for targeting and replacement of other genomic sequences, and could be a useful approach to repurpose existing transgenes as new genetic reagents become available.
ISSN:1943-2631
0016-6731
1943-2631
DOI:10.1534/genetics.116.191783