A gene-specific library for .

We generated a library of ~1000 stocks in which we inserted a construct in the intron of genes allowing expression of under control of endogenous promoters while arresting transcription with a polyadenylation signal 3' of the GAL4. This allows numerous applications. First, ~90% of insertions in essential genes cause a severe loss-of-function phenotype, an effective way to mutagenize genes. Interestingly, 12/14 chromosomes engineered through CRISPR do not carry second-site lethal mutations. Second, 26/36 (70%) of lethal insertions tested are rescued with a single cDNA construct. Third, loss-of-function phenotypes associated with many insertions can be reverted by excision with . Fourth, driven reports tissue and cell-type specificity of gene expression with high sensitivity. We report the expression of hundreds of genes not previously reported. Finally, inserted cassettes can be replaced with or any DNA. These stocks comprise a powerful resource for assessing gene function.