Pervasive Cas9 expression driven by mammalian promoter activity in E. coli affects representation of CRISPR sgRNA...

CRISPR-Cas9 screening relies on uniform representation of single-guide RNA (sgRNA) libraries to enable accurate gene discovery. However, technical biases during library preparation can compromise screen performance. Here we show that commonly used "all-in-one" CRISPR vectors expressing both Cas9 and sgRNAs drive unintended Cas9 protein expression in Escherichia coli during plasmid amplification. This bacterial Cas9 expression causes guide-specific toxicity, leading to selective loss of sgRNAs and highly skewed library representation. We demonstrate that this effect occurs across multiple bacterial strains and affects both targeted and genome-wide libraries, including widely used human CRISPR libraries. Mechanistically, toxicity is driven by Cas9 expression rather than plasmid size and is only partially alleviated by catalytically inactive Cas9. Importantly, replacing the EF-1α promoter with a mouse phosphoglycerate kinase promoter suppresses Cas9 expression in bacteria while preserving genome editing efficiency in mammalian cells, restoring sgRNA uniformity. These findings identify a previously unrecognized source of bias in CRISPR library preparation and provide a practical solution to improve screening fidelity.