Transcription modulation of pathogenic streptococcal and enterococcal species using CRISPRi technology

Enterococcus faecalis is a gram-positive opportunistic pathogen known to cause multidrug-resistant infections including sepsis, endontic infections, and hospital-acquired urinary tract infections [8–10]. E. faecalis possess a type-II CRISPR-Cas9 system. Interestingly, E. faecalis clinical isolates that are multidrug-resistant have mobile genetic elements that can be targeted by CRISPR-Cas systems and then maintained [11]. E. faecalis has shown that there is a phenotype of “CRISPR tolerance,” which corresponds to transient maintenance of targeted DNA [11].

Targeted mutagenesis through allelic exchange has been a frequently used method for genome editing. An alternative to this method was developed in E. faecalis, which utilizes a two-plasmid CRISPR-Cas9-mediated genome editing system that allows for a reduction of screening needed, as well as a shortened period for genome editing [12]. Another two-plasmid system was generated in Enterococcus faecium, utilizing inducible expression of RecT, an ssDNA-annealing protein associated with phage recombination, in combination with CRISPR-Cas9-based counterselection. This system permits either genomic insertion or deletion while minimizing screening, making it a rather versatile approach to mutagenesis [13]. Limitations of this system include reduced transformation efficiency due to the toxicity of Cas9, as well as the need for multiple large vectors [14].

An E. faecalis CRISPRi tool was created for use in high-throughput, scalable genetic control studies eliminating the limitation around reduced transformation results with the genome editing system. A dual-vector nisin-inducible system was tested and shown to silence both single genes and whole operons [15]. Additionally, the system was able to silence genes involved in biofilm formation and antibiotic resistance [15]. Future applications include investigating antibiotic resistance mechanisms and virulence factors associated with E. faecalis infection.

S. pyogenes is a gram-positive bacterium responsible for many infections including pharyngitis, necrotizing fasciitis, and toxic shock syndrome [16–18]. S. pyogenes can possess either CRISPR Type I-C or Type II-A.

S. pyogenes is notable for the Cas9 system known as Spy Cas9, which was utilized to develop CRISPRi [19]. The process entailed cloning the S. pyogenes Cas9 gene along with a minimal CRISPR locus into either an expression vector or the target organism’s genome. Spy Cas9 has been used extensively as a platform for producing CRISPRi functionality in bacteria that do not harbor an endogenous Cas9 [5,20].

While Spy Cas9 has been used to apply CRISPRi in other species, the same could not be said for S. pyogenes until recently. A study has shown successful implementation of a doxycycline-inducible dCas9-based CRISPRi system in S. pyogenes [21]. The doxycycline induction system permits tight expression regulation, and researchers were able to demonstrate that the system could reliably knock down target genes of interest. A library of unique spacers featuring near-complete coverage of the S. pyogenes genome was made accessible in SpyBrowse, the S. pyogenes genome browser. Future directions include the transformation and implementation of the spacers, and generation of a CRISPRi knockdown library.

Additionally, it has been demonstrated that in S. pyogenes strains with a type II-A CRISPR-Cas system, there are fewer prophages compared to type I-C CRISPR-Cas strains [22]. Interestingly, it was also shown that a unique streptococcal phage “anti-CRISPR,” a phage-encoded protein that interferes with CRISPR-Cas systems, can inhibit S. pyogenes Cas9 [23]. Future studies are needed to examine the role of anti-CRISPRs in other streptococcal species.

S. agalactiae is a gram-positive pathobiont that colonizes the genitourinary and gastrointestinal mucosa. S. agalactiae is capable of opportunistic vertical transmission to the neonate, which can result in meningitis, chorioamnionitis, or neonatal sepsis [24,25]. Additionally, S. agalactiae is also a rare cause of infection in nonpregnant adults [26,27]. S. agalactiae, like S. pyogenes, harbors either a Type-I-C or Type-II-A CRISPR system, which has been exploited to produce CRISPRi strains like those generated in S. pyogenes. Notably, the residues associated with Cas9 catalytic activity do vary from S. pyogenes, with the HNH nuclease domain shifted such that the point mutation associated with catalytic inactivation is H845A rather than H840A [19,28].

CRISPRi has been applied as a tool for the generation of rapid, flexible knockdown phenotypes [28,29]. CRISPRi has also been utilized to investigate the various roles that the CRISPR/Cas system plays apart from its canonical role as a nuclease, highlighting a potential role in regulating virulence, which has been suggested in previous non-CRISPRi studies [28,30]. There has been a recent surge in the development of CRISPRi technologies for S. agalactiae. A group has developed multiple dCas9 strains for use in exploring S. agalactiae transcription and fitness [28,29]. These developments can be expanded on for future research, with the potential for added complexity using sgRNA multiplexing.

S. pneumoniae is a gram-positive pathobiont colonizing the upper respiratory mucosa that can cause pneumonia, meningitis, or otitis media [31]. S. pneumoniae does not possess an endogenous CRISPR/Cas system, but the natural competence and relative ease of genetic manipulation simplifies most CRISPRi-based applications. One common approach is the chromosomal insertion of Spy dCas9 and a single-guide RNA (sgRNA), allowing for expression. Additionally, this system can be driven by an inducible promoter, allowing for greater experimental complexity [32,33].

In S. pneumoniae, CRISPRi has been applied in combination with high-throughput Tn-seq (transposon sequencing) [34]. Tn-seq-based essential gene identification can be followed up with CRISPRi screening to identify previously unexplored genes. Tn-seq has been utilized in S. pneumoniae strain D39 for the identification of genes potentially related to antibiotic resistance [33]. Another application of CRISPRi is CRISPRi-seq and its notable subtype sCRilecs-seq. CRISPRi-seq is an approach that allows for minimal off-target effects through careful targeting of all operons rather than individual genes [32]. sCRilecs-seq (subsets of CRISPR interference libraries extracted by fluorescence-activated cell sorting coupled to next-generation sequencing) is a process that couples CRISPRi with fluorescence-activated cell sorting (FACS) to identify loci associated with a given phenotype [35]. Bacteria with desired fluorescence intensities can be collected and subject to sequencing to determine identity. This technique has been used to identify pathways that could function as combination treatment targets to deal with the growing threat of ß-lactam resistance in S. pneumoniae [36]. The ease of mutagenesis in S. pneumoniae facilitates the utilization of complex screening techniques, allowing CRISPRi technology to continue to expand and provide valuable results.

CRISPRi is a growing technology, with recent developments across different bacteria. The strengths of CRISPRi motivate continued development of screening technology as rapid, targeted expression manipulation is highly versatile. The push to develop CRISPRi technology in S. agalactiae is notable, yielding insights into transcription and fitness. Additionally, advanced applications such as SCRilecs-seq and CRISPRi-seq in S. pneumoniae or the nisin-inducible system developed for E. faecalis allow for increasingly complex and specialized investigations into antibiotic resistance mechanisms and virulence associated genes [15,32].

A future direction of interest would be utilization of Mobile CRISPRi, a technique developed for Pseudomonas aeruginosa. Mobile-CRISPRi allows for the transfer and integration of CRISPRi into bacteria through an antibiotic resistance cassette, sgRNA spacers, and a promoter that drives dCas9 expression [37]. CRISPRi technology is expanding and continues to demonstrate exciting results as it is used in a wider span of bacterial species. Additionally, CRISPRi may be of significant value to agricultural fields requiring scalable and functional investigation into target genes [38,39].