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

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.