Monday, November 25, 12:30pm – 12:50pm, Zeis 123, via Zoom
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Jacob Fender
Dr. Melinda Grosser
CRISPR interference (CRISPRi) is a technique that utilizes a catalytically “dead” Cas9 complex to sterically block the transcription of a gene targeted by a guide RNA. CRISPRi can be used for knockdown and subsequent study of essential genes in bacteria, especially those lacking corresponding mutants in commercial transposon mutant libraries. This project optimized and tested an inducible CRISPRi system that we designed for the knockdown of essential genes in Staphylococcus aureus, which was then used by undergraduate students in an upper-level Molecular Biology lab course. Our lab’s previous attempts to optimize a CRISPRi system were hindered by supercoiling and extremely inefficient restriction enzyme digestion of the large plasmid (pRMC2-CRISPRi) which had been designed as a vector, following its extraction from its E. coli host (strain Dh5-alpha). After switching to a methylase-negative strain of E. coli as a vector host, digestion of miniprepped pRMC2-CRISPRi plasmids with BsaI had greatly increased efficiency, with no undigested vector remaining visible. This was unexpected, as there is not a predicted methylation site near the BsaI restriction sites. Guide RNAs targeting several essential or nearly essential genes were ligated into the vector via Golden Gate assembly, confirmed via colony PCR and sequencing. The completed plasmids were transformed into S. aureus RN4220 and subsequently into the community-acquired MRSA strain LAC. Knockdown of the target genes was tested in growth assays, where induction of the CRISPRi system causes at least partial growth inhibition. Future work will include qRT-PCR verification of all knockdown strains in LAC and further phenotypic testing, especially for knockdowns of genes with unknown functions.