Tools and strategies for scarless allele replacement in Drosophila using CRISPR/Cas9

• Published in: Fly (Austin, Tex.) Vol. 11; no. 1; pp. 53 - 64
• Main Authors: Lamb, Abigail M., Walker, Elizabeth A., Wittkopp, Patricia J.
• Format: Journal Article
• Language: English
• Published: United States Taylor & Francis 02.01.2017; Taylor & Francis Group
• Subjects: Alleles; Animals; Animals, Genetically Modified; crispr; CRISPR-Cas Systems; Drosophila melanogaster; Drosophila melanogaster - genetics; Drosophila melanogaster - growth & development; Drosophila Proteins - antagonists & inhibitors; Drosophila Proteins - genetics; Drosophila Proteins - metabolism; gene editing; Gene Editing - methods; genetic analysis; genetic background; genome editing; homology directed repair; Methods and Technical Advances; Mutation; non-model species; point mutation; pupae; screening; single-nucleotide; transgenic; wings; CRISPR; genome editing; homology directed repair; single-nucleotide; transgenic; non-model species; point mutation
• ISSN: 1933-6934; 1933-6942; 1933-6942
• DOI: 10.1080/19336934.2016.1220463

Genome editing via the CRISPR/Cas9 RNA-guided nuclease system has opened up exciting possibilities for genetic analysis. However, technical challenges associated with homology-directed repair have proven to be roadblocks for producing changes in the absence of unwanted, secondary mutations commonly known as "scars." To address these issues, we developed a 2-stage, marker-assisted strategy to facilitate precise, "scarless" edits in Drosophila with a minimal requirement for molecular screening. Using this method, we modified 2 base pairs in a gene of interest without altering the final sequence of the CRISPR cut sites. We executed this 2-stage allele swap using a novel transformation marker that drives expression in the pupal wings, which can be screened for in the presence of common eye-expressing reporters. The tools we developed can be used to make a single change or a series of allelic substitutions in a region of interest in any D. melanogaster genetic background as well as in other Drosophila species.