Jiang, W. et al. Cell 180, 1002–1017 (2020).

Genome-wide CRISPR screens are a powerful tool for revealing gene functions. Yet the synthesis of guide RNA libraries can be expensive and time consuming. Jiang et al. leverage naturally occurring CRISPR–Cas adaptation machinery to convert exogenous DNA into CRISPR RNA (crRNA) libraries in bacteria. More specifically, they transform Staphylococcus aureus cells with sheared genomic DNA of interest via electroporation, which generates genome-wide crRNA libraries. These libraries can be subcloned into other species or directly used in S. aureus; the latter circumvents cloning and transformation steps. When cells were electroporated with Escherichia coli DNA, the resulting library covered over 4,000 genes with an average of 103 crRNAs per gene. In a CRISPR interference screen, the crRNA libraries were subcloned into E. coli and used to survey a range of transcriptional repression, leading to an identification of new antibiotic-potentiating pathways.