PgmNr D1519: Efficient targeted editing of genes with a modified Crispr/Cas9 strategy
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Authors:
D. Li-Kroeger 1,3 ; Y. He 3,4 ; H. Bellen 1,2,3,4


Institutes
1) Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 2) Department of Neuroscience, Baylor College of Medicine, Houston, TX; 3) Neurological Research Institute, Houston, Texas; 4) HHMI, Baylor College of Medicine.


Keyword: gene targeting and modification

Abstract:

Here we present a Knock-in/Knock out approach to Crispr/Cas9 gene editing in Drosophila that allows simple dominant marker-based screening to delete DNA regions of interest.  The insert cassette is engineered to allow precise and efficient replacement of the deleted segment with any DNA, allowing scarless swapping of engineered sequences for downstream applications.  We created a modular toolkit for tagging any gene.  We demonstrate the utility of these tools by creating dominantly marked loss-of-function alleles of several genes including the neuronal maintenance factor Nicotinamide Adenylyl Transferase (Nmnat).  We then show using the Nmnat allele that we can efficiently swap the inserted cassette by creating Nmnat:GFP:Nmnat fusion proteins with either wild-type or mutated sequences.  We find using multiple replicates of injection that greater than fifteen percent of vials from injected (G0) larvae contain Knock-in genes with seamless cassette swapping. In summary, we have created a toolkit and supporting reagents that allow genomic editing in Drosophila and permits scarless modification of regions of interest for downstream applications.