Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function

Abstract

A recent genome-wide screen identified~300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9 Smu) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9 Smu with a constitutively active singleguide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (Cas9 Spy). We then investigated sgRNA specificity and proto-spacer adjacent motif (PAM) requirements. Interference by sgRNAs did not occur with double or triple base-pair mutations, or if single base-pair mutations were in the 3' end of the sgRNA. Bioinformatic analysis of >450 S. mutans genomes allied with in vivo assays revealed a similar PAM recognition sequence as Cas9 Spy. Next, we created a comprehensive library of sgRNA plasmids that were directed at essential and growth-supporting genes. We discovered growth defects for 77% of the CRISPRi strains expressing sgRNAs. Phenotypes of CRISPRi strains, across several biological pathways, were assessed using fluorescence microscopy. A variety of cell structure anomalies were observed, including segregational instability of the chromosome, enlarged cells, and ovococci-to-rod shape transitions. CRISPRi was also employed to observe how silencing of cell wall glycopolysaccharide biosynthesis (rhamnose-glucose polysaccharide, RGP) affected both cell division and pathogenesis in a wax worm model. The CRISPRi tool and sgRNA library are valuable resources for characterizing essential genes in S. mutans, some of which could prove to be promising therapeutic targets.

Morphological and Transcriptional Responses to CRISPRi Knockdown of Essential Genes in Escherichia coli

“…1C ). Only 53 essential gene knockdown strains exhibited a fitness defect ≥10% (RF < 0.9), indicating that many essential gene products are present in excess in wild-type cells, likely to buffer against environmental fluctuations, as previously suggested by pooled and arrayed approaches in E. coli , B. subtilis , and other species ( 8 , 10 , 15 , 18 , 20 ).…”

“…Arrayed libraries of CRISPRi strains targeting essential genes have been of particular utility, because they enable flexible pooling and candidate follow-ups of pooled assays as well as single-strain assays such as microscopy. Such libraries have been described for Bacillus subtilis ( 8 ), Streptococcus pneumoniae ( 9 ), Streptococcus mutans ( 10 ), and Mycobacterium smegmatis ( 11 ) and have been used to yield surprising cross-pathway functional interactions, insights into cellular vulnerabilities, and functional characterizations of essential genes. Remarkably, no such library has been described for any Gram-negative bacterium despite the original demonstration of CRISPRi in Escherichia coli ( 7 ), the veritable cornucopia of pooled CRISPRi studies in Gram-negative bacteria (reviewed in reference 12 ), and the significant differences between Gram-negative and Gram-positive physiology centered around essential functions such as the Gram-negative-specific outer membrane.…”

Characterization of FtsH Essentiality in Streptococcus mutans via Genetic Suppression

“…Furthermore, as shown in Figure 1D , the growth of strain pXylS1 ftsH /∆ ftsH is critically dependent on the presence of xylose in the medium, which confirmed the essentiality of FtsH in S. mutans . This is also consistent with recent Tn-seq and CRISPRi screening studies in S. mutans , which suggested ftsH to be essential ( Shields et al, 2018 , 2020 ).…”

Manganese Depletion Leads to Multisystem Changes in the Transcriptome of the Opportunistic Pathogen Streptococcus sanguinis

“…The last phosphatase, PpaC, is essential for S. sanguinis (Xu et al, 2011), so if PpaC activity was decreased due to Mn depletion, this could have contributed to the decreased growth rate phenotype observed post-EDTA. Further studies utilizing the knockout mutants of each non-essential phosphatase (Xu et al, 2011) or an approach such as CRISPR interference (Shields et al, 2020) for PpaC would enhance our understanding of the relative contributions of each phosphatase to the growth and morphology of S. sanguinis.…”