First test of stock CFD-5: UAS-cas9

Hui-Min Chen and Tzumin Lee

Janelia Farm Research Campus, HHMI

 

We expressed UAS-cas9 (stock CDF-5) under the control of a germline Gal4 line and crossed these flies to transgenic flies expressing gRNAs targeting yellow under the control of the U6-3 promoter (pU6-3-gRNA y). The gRNA target site was: GGAGCGATATAGTTGGAGCCAGCTGG. Female offspring of this cross was then crossed to yellow mutant males and yellow mutant offspring was counted.

Of the 10 females we crossed 4 gave yellow mutant offspring (40% founder animals). The number of mutant offspring was 1/180 (0.5%), 2/245 (0.8%), 3/84 (3.6%) and 1/117 (0.9%).

Interestingly, a similar experiment using a UAS-cas9 transgene with only 3 UAS site (as compared to 10 UAS sites used for CFD-5) did not yield any yellow mutant offspring, suggesting that cas9 levels are limiting. Furthermore, use of the U6-2 promoter to drive gRNA expression also resulted in no targeting. We are currently testing more combinations of cas9 transgenes and U6 promoters to get an idea what the best combinations are.