An inducible CRISPR-interference library for genetic interrogation of Saccharomyces cerevisiae biology

Genome-scale CRISPR interference (CRISPRi) is widely utilized to study cellular processes in a variety of organisms. To date, a genome-wide CRISPRi library, optimized for targeting the Saccharomyces cerevisiae genome, has not been presented. Here, we have generated a comprehensive, inducible CRISPRi library, based on spacer design rules optimized for yeast. We have validated this library for genome-wide interrogation of gene function across a variety of applications, including accurate discovery of haploinsufficient genes and identification of enzymatic and regulatory genes involved in adenine and arginine biosynthesis. The comprehensive nature of the library also revealed refined spacer design parameters for transcriptional repression, including location, nucleosome occupancy and nucleotide features. CRISPRi screens using this library can identify genes and pathways with high precision and low false discovery rate across a variety of experimental conditions, enabling rapid and reliable genome-wide assessment of genetic function and interactions in S. cerevisiae. We designed more than 51,000 gRNAs, with between 6-12 sequences per gene. As a negative control for gRNA activity, we introduced 500 synthetic randomly shuffled gRNAs with no matches in the yeast genome. We generated two independently cloned bacterial library replicates. The two bacterial biological replicates libraries were transformed into yeast.