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Multilayered regulation by RNA thermometers enables precise control of Cas9 expression in E. coli

ABSTRACT: Cas9-based genome editing technologies can rapidly generate mutations to probe a diverse array of mutant genotypes. However, aberrant Cas9 nuclease translation and activity can occur despite the use of inducible promoters to control expression, leading to extensive cell death. This background killing caused by promoter leakiness severely limits the application of Cas9 for generating mutant libraries because of the potential for population skew. We demonstrate the utility of temperature sensitive RNA elements as a layer of post-transcriptional regulation to reduce the impact of promoter leak. We observe significant temperature-dependent increases in cell survival when certain RNA thermometers (RNATs) are placed upstream of the cas9 coding sequence. We also show that the most highly repressing RNAT, hsp17rep, significantly reduces population skew with a library of characterized guide RNAs in Escherichia coli. This strategy should be applicable to all bacterial Cas9-based methods and technologies.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Escherichia Coli (ncbitaxon:562)

SUBMITTER: William G. Alexander

PROVIDER: MSV000100641 | MassIVE | Thu Jan 29 06:54:00 GMT 2026

SECONDARY ACCESSION(S): PXD073781

REPOSITORIES: MassIVE

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