title: Efficient Screening of CRISPR/Cas9-Induced Events in Drosophila Using a Co-CRISPR Strategy.
abstract: Genome editing using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated nuclease (Cas9) enables specific genetic modifications, including deletions, insertions, and substitutions in numerous organisms, such as the fruit fly Drosophila melanogaster One challenge of the CRISPR/Cas9 system can be the laborious and time-consuming screening required to find CRISPR-induced modifications due to a lack of an obvious phenotype and low frequency after editing. Here we apply the successful co-CRISPR technique in Drosophila to simultaneously target a gene of interest and a marker gene, ebony, which is a recessive gene that produces dark body color and has the further advantage of not being a commonly used transgenic marker. We found that Drosophila broods containing higher numbers of CRISPR-induced ebony mutations ("jackpot" lines) are significantly enriched for indel events in a separate gene of interest, while broods with few or no ebony offspring showed few mutations in the gene of interest. Using two different PAM sites in our gene of interest, we report that ∼61% (52-70%) of flies from the ebony-enriched broods had an indel in DNA near either PAM site. Furthermore, this marker mutation system may be useful in detecting the less frequent homology-directed repair events, all of which occurred in the ebony-enriched broods. By focusing on the broods with a significant number of ebony flies, successful identification of CRISPR-induced events is much faster and more efficient. The co-CRISPR technique we present significantly improves the screening efficiency in identification of genome-editing events in Drosophila.
authors: Waksman Institute, Department of Molecular Biology and Biochemistry, Cancer Institute of New Jersey, Rutgers University, Piscataway, New Jersey 08854-8020.
authors: Nanci S
authors: NS
authors: Kane
authors: Waksman Institute, Department of Molecular Biology and Biochemistry, Cancer Institute of New Jersey, Rutgers University, Piscataway, New Jersey 08854-8020.
authors: Mehul
authors: 0000-0002-1239-6458
authors: M
authors: Vora
authors: Waksman Institute, Department of Molecular Biology and Biochemistry, Cancer Institute of New Jersey, Rutgers University, Piscataway, New Jersey 08854-8020.
authors: Krishna J
authors: KJ
authors: Varre
authors: Waksman Institute, Department of Molecular Biology and Biochemistry, Cancer Institute of New Jersey, Rutgers University, Piscataway, New Jersey 08854-8020 padgett@waksman.rutgers.edu.
authors: Richard W
authors: 0000-0002-5740-3324
authors: RW
authors: Padgett
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D064113:CRISPR-Cas Systems / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D029721:Drosophila Proteins / Q000235:genetics*; D004331:Drosophila melanogaster / Q000235:genetics*; D000072669:Gene Editing / Q000379:methods*; D018390:Gene Targeting / Q000379:methods; D054643:INDEL Mutation / Q000235:genetics; D010641:Phenotype
chemical_list: D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C116191:e protein, Drosophila
keywords: CRISPR; Cas9; Drosophila; co-CRISPR; co-conversion