Simplified homology-assisted CRISPR for gene editing in Drosophila

• Published in: G3 : genes - genomes - genetics 2024-02, Vol.14 (2)
• Main Authors: Rankin, Anne E, Fox, Elizabeth, Chisholm, Townley, Lantz, Nicole, Rajan, Arjun, Phillips, William, Griffin, Elizabeth, Harper, Jaekeb, Suhr, Christopher, Tan, Max, Wang, Jason, Yang, Alana, Kim, Ella S, Ankrah, Naa Kwama A, Chakraborty, Praachi, Lam, Alistair C K, Laws, Madeleine E, Lee, Jackson, Park, Kyle K, Wesel, Emily, Covert, Peter H, Kockel, Lutz, Park, Sangbin, Kim, Seung K
• Format: Article
• Language: English
• Subjects: Investigation
• ISSN: 2160-1836; 2160-1836
• DOI: 10.1093/g3journal/jkad277

Abstract In vivo genome editing with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 generates powerful tools to study gene regulation and function. We revised the homology-assisted CRISPR knock-in method to convert Drosophila GAL4 lines to LexA lines using a new universal knock-in donor strain. A balancer chromosome–linked donor strain with both body color (yellow) and eye red fluorescent protein (RFP) expression markers simplified the identification of LexA knock-in using light or fluorescence microscopy. A second balancer chromosome–linked donor strain readily converted the second chromosome–linked GAL4 lines regardless of target location in the cis-chromosome but showed limited success for the third chromosome–linked GAL4 lines. We observed a consistent and robust expression of the yellow transgene in progeny harboring a LexA knock-in at diverse genomic locations. Unexpectedly, the expression of the 3xP3-RFP transgene in the “dual transgene” cassette was significantly increased compared with that of the original single 3xP3-RFP transgene cassette in all tested genomic locations. Using this improved screening approach, we generated 16 novel LexA lines; tissue expression by the derived LexA and originating GAL4 lines was similar or indistinguishable. In collaboration with 2 secondary school classes, we also established a systematic workflow to generate a collection of LexA lines from frequently used GAL4 lines.