Using CRISPRi in Escherichia coli to emphasize experimental controls in a molecular microbiology laboratory

A CRISPR interference (CRISPRi) exercise was developed for an upper-level molecular microbiology laboratory to reinforce student skills in experimental design and controls. This CRISPRi knockdown method is a variation of the commonly used Streptococcus pyogenes Cas9 system and therefore relies on similar design and techniques. Students choose and design a CRISPRi target in Escherichia coli, clone the necessary tools, and test their system with spot plating and microscopy. The motivation for introducing this unit in a laboratory course was to help close gaps in students' broader understanding of DNA and RNA structure, primer design, bacterial gene expression, and regulation. Once introduced, this exercise became a way to help students identify, design, and rationalize proper experimental controls.