Identifying core and accessory essential genes of genetically diverse Staphylococcus epidermidis strains with genome-wide CRISPRi screens
In: Student Reports, Summer 2023, The Jackson Laboratory
Elizabeth Fleming, Georgia Doing, Ph.D. and Julia Oh, Ph.D.
The high strain diversity and versatility of Staphylococcus epidermidis allows it to play a dual role in the skin microbiome, either conferring protection against pathogens or acting as a pathogen itself. Key to understanding what makes a pathogenic or commensal strain of S. epidermidis is characterizing the accessory genome, consisting of genes that are unique to one or a few strains. CRISPR interference (CRISPRi) facilitates the study of gene function and can be scaled genome- wide through the customization of guideRNA sequences to target each gene in the genome. However, the requirement of an NGG PAM site for this technique leaves some genes inaccessible, most notably in genomes with low GC content like S. epidermidis. The development of PAM-less Cas9 variants, such as SpRYc, have the potential to expand the scope of CRISPR- Cas9 targeting. Though we have not yet demonstrated effective knockdown with dSpRYc in a CRIPSRi context, genome-wide PAM-less guide design with GuideFinder increases the range of targetable genes across ten strains of S. epidermidis to greater than 96% with NAN or NNN PAM sites and up to 99.2% under less stringent filtering thresholds. We also design a genome-wide CRISPRi screen of strain NIHLM031 for identification of essential genes. Despite limitations, PAM-less CRISPRi targeting is promising for expanding genome-wide screens and providing insight to core and accessory genes of S. epidermidis.
Mellor, Sierra, "Identifying core and accessory essential genes of genetically diverse Staphylococcus epidermidis strains with genome-wide CRISPRi screens" (2023). Summer and Academic Year Student Reports. 2736.