title: Tools and strategies for scarless allele replacement in Drosophila using CRISPR/Cas9.
abstract: 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.
authors: a Department of Molecular, Cellular, and Developmental Biology , University of Michigan , Ann Arbor , MI , USA.
authors: Abigail M
authors: AM
authors: Lamb
authors: b Department of Ecology and Evolutionary Biology , University of Michigan , Ann Arbor , MI , USA.
authors: Elizabeth A
authors: EA
authors: Walker
authors: a Department of Molecular, Cellular, and Developmental Biology , University of Michigan , Ann Arbor , MI , USA.|b Department of Ecology and Evolutionary Biology , University of Michigan , Ann Arbor , MI , USA.
authors: Patricia J
authors: PJ
authors: Wittkopp
mesh_terms: D000483:Alleles; D000818:Animals; D030801:Animals, Genetically Modified; D064113:CRISPR-Cas Systems; D029721:Drosophila Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics* / Q000254:growth & development; D000072669:Gene Editing / Q000379:methods*; D009154:Mutation
chemical_list: D029721:Drosophila Proteins
keywords: CRISPR; genome editing; homology directed repair; non-model species; point mutation; single-nucleotide; transgenic