Project Summary
Streptococcus agalactiae (Group B Streptococcus, GBS) is an asymptomatic colonizer of the genital and
gastrointestinal tracts of healthy people, however, it can become a major cause of disease especially in
immunocompromised individuals and the neonate. GBS is the leading cause of neonatal meningitis and
presently there is no vaccine available. Therefore, there is much interest in discovering how GBS is able to
colonize a healthy host, interact with other microbes in the mucosa, and cause invasive disease. For years,
researchers have relied on non-targeted mutagenesis strategies such as transposon mutagenesis to discover
novel GBS virulence factors. However, these strategies have the potential to miss the impact of essential
genes, can be prone to “hotspots”, and are not ordered. Recently, CRISPR interference (CRISPRi)
technologies have enabled researchers to investigate the contribution of any gene of interest rapidly by
generating a targeted knockdown. The aim of this proposal is to generate an ordered CRISPRi library in the
hypervirulent strain of GBS, COH1 (Type III, MLST-17). First, we will generate a deactivated Cas9 mutant in
GBS that is required for CRISPRi. Next, we will generate two-fold coverage knockdowns of each gene in the
GBS COH1 genome, index, and cryopreserve. The generated library will be made available to the wider GBS
community and can be utilized to investigate aspects of GBS colonization and pathogenesis such as host-
pathogen interactions, GBS colonization of gut and genital tract mucosal environments, microbe-microbe
interactions, immune evasion, and many more. Additionally, the library will be thoughtfully designed so that
guide RNA constructs, where applicable, will be effective in other commonly used laboratory strains of GBS.
Therefore our proposed library will provide not only the COH1 knockdown library to the field, but also a library
of guide RNA knockdown constructs amenable to the wider GBS community. Using the CRISPRi library, we
anticipate to propel the GBS field forward that could uncover novel interactions between the host and pathogen
that could lead to therapeutic interventions.
