Transcription modulation of pathogenic streptococcal and enterococcal species using CRISPRi technology

CRISPR, short for clustered regularly interspaced short palindromic repeats, is found in 40% of bacteria and 85% of archaea providing defense against phage and other foreign DNA [1-3].The CRISPR locus contains both "spacers," which encode guide RNAs (gRNAs) used to direct the system to target nucleic acids, and Cas effector proteins.There are several Cas effector proteins with some functioning as endonucleases, while others serve for recognition or processing of gRNA.Certain Cas effectors, such as Cas9, can be directed to cut a given site via recognition of a gRNA, creating a double-stranded break in the target DNA.These effectors can scan long sequences of DNA for short sequences called protospacer adjacent motifs (PAMs), before unraveling the DNA to cut.These PAMs vary between different CRISPR-Cas types [4,5].CRISPR interference (CRISPRi) utilizes a catalytically dead Cas effector (dCas), through targeted mutation of the catalytic domains, specifically HNH and RuvC-like nuclease domains in Cas9, to eliminate Cas endonuclease activity [4,5].This dCas effector retains its ability to recognize a gRNA and bind DNA.If guided to a gene, dCas will bind and form a "roadblock" on the gene, reducing transcription and causing a knockdown in expression of the gene as shown in Fig 1.This can be most easily achieved in Class 2 Cas effectors (primarily Cas9 and Cas12a), as these rely primarily on a single effector for both target binding and DNA cleavage rather than the effector complexes of Class 1 systems, which feature a cascade of Cas proteins (shown in Fig 2) [6,7].dCas transcriptional regulators allow for near-silencing of multiple genes making CRISPRi technology robust and high throughput.CRISPRi systems can be modified and applied to a variety of bacteria.We will review recent examples of CRISPRi technology in Enterococcus spp., Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus pneumoniae.We will also discuss future applications of CRISPRi technology including direct genome editing, gene silencing, and CRISPRseq.