Simplified homology-assisted CRISPR for gene editing in Drosophila

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.

CRISPR/Cas9 gene editing inDrosophilavia visual selection in a summer classroom

“…We postulated that the HACK (Homology Assisted CRISPR Knock-In; Lin and Potter, 2016) crossing scheme using the visible selectable marker yellow + (Rankin et al, 2023) could be adapted for use in an accelerated summer classroom setting lasting seven weeks ( Figure 1A, B ). Twelve neuropeptide- Gal4 lines from the Bloomington Drosophila Stock Center (BDSC; Methods) were selected to convert to LexA driver lines in the 2023 “Animal Transgenesis” course at Harvard Summer School with 12 participants.…”

“…Twelve neuropeptide- Gal4 lines from the Bloomington Drosophila Stock Center (BDSC; Methods) were selected to convert to LexA driver lines in the 2023 “Animal Transgenesis” course at Harvard Summer School with 12 participants. The ‘version 2’ HACKing donor for inter -chromosomal conversion, HACKy.V2 located on the CyO chromosome ( Figure 1 ; “CyO HACKy.V2,y+RFP+ ” donor hereafter), was reported to show optimized results for HACKing of second chromosome locations (Rankin et al, 2023), and the selected Gal4 lines were reported to reside on chromosome II by their associated data in the BDSC database (see below).…”

“…Additional testing for the co-expression of the RFP + eye marker in yellow + conversion males showed both selectable markers were consistently present ( Figure 2 ). Eye expression of RFP was driven by a pax6 promoter element (Rankin et al, 2023; Sheng et al, 1997). In summary, within seven weeks, students successfully applied CRISPR targeting, genetics, and marker selection (Rankin et al, 2023) to derive multiple novel LexA lines.…”

“…y 1 , w * , vas-Cas9; CyO HACKy . V2,y+,RFP+ /L , and y,w; CyO/L; TM2/TM6B were previously described (Rankin et al, 2023).…”

“…In prior work, the identification of successfully ‘HACKed’ Gal4 -to- LexA convertants required fluorescence microscopy (Lin and Potter, 2016). A modified and successful approach to convert existing Gal4 lines into LexA drivers was recently described as a convenient tool-kit suitable for an 11-week secondary school class (Rankin et al, 2023); in that work, integration of a yellow + cassette as a selectable marker gene permitted brightfield microscopy-based methods to identify successful Gal4 -to- LexA convertants. We postulated that this technical innovation could broaden use of this experimental approach (Rankin et al, 2023).…”