We generated a library of ~1,000stocks in which we inserted a construct in the intron of genes allowing expression ofunder 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 singlecDNA construct. Third, loss-of-function phenotypes associated with manyinsertions can be reverted by excision with. Fourth,drivenreports 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 withor any DNA. These stocks comprise a powerful resource for assessing gene function.