Rankin, A.E., Fox, E., Chisholm, T., Lantz, N., Rajan, A., Phillips, W., Griffin, E., Harper, J., Suhr, C., Tan, M., Wang, J., Yang, A., Kim, E.S., Ankrah, N.K.A., Chakraborty, P., Lam, A.C.K., Laws, M.E., Lee, J., Park, K.K., Wesel, E., Covert, P.H., Kockel, L., Park, S., Kim, S.K. (2024). Simplified homology-assisted CRISPR for gene editing in Drosophila.  G3 (Bethesda) 14(2): jkad277.

FBrf0258754

Research paper

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.

PMC10849607 (PMC) (EuropePMC)