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CRISPR-Cas systems are prokaryotic adaptive immune systems that recognize and cleave nucleic acid targets using small RNAs called CRISPR RNAs (crRNAs) to guide Cas protein(s). There is increasing evidence for the broader endogenous roles of these systems. The CRISPR-Cas9 system of Francisella novicida also represses endogenous transcription using a non-canonical small RNA (scaRNA). We examined whether the crRNAs of the native F. novicida CRISPR-Cas systems, Cas12a and Cas9, can guide transcriptional repression. Both systems repressed mRNA transcript levels when crRNA-target complementarity was limited, and led to target cleavage with extended complementarity. Using these parameters we engineered the CRISPR array of Cas12a to guide the transcriptional repression of a new and endogenous target. Since the majority of crRNA targets remain unidentified, this work suggests that a re-analysis of crRNAs for endogenous targets with limited complementarity could reveal new, diverse regulatory roles for CRISPR-Cas systems in prokaryotic biology.

CRISPR-Cas systems are prokaryotic adaptive immune systems that facilitate protection of bacteria and archaea against infection by external mobile genetic elements. The model pathogen Francisella novicida encodes a CRISPR-Cas12a (FnoCas12a) system and a CRISPR-Cas9 (FnoCas9) system, the latter of which has an additional and noncanonical function in bacterial virulence. Here, we investigated and compared the functional roles of the FnoCas12a and FnoCas9 systems in transformation inhibition and bacterial virulence. Unlike FnoCas9, FnoCas12a was not required for F. novicida virulence. However, both systems were highly effective at plasmid restriction and acted independently of each other. We further identified a critical protospacer-adjacent motif (PAM) necessary for transformation inhibition by FnoCas12a, demonstrating a greater flexibility for target identification by FnoCas12a than previously appreciated and a specificity that is distinct from that of FnoCas9. The effectors of the two systems exhibited different patterns of expression at the mRNA level, suggesting that they may confer distinct benefits to the bacterium in diverse environments. These data suggest that due to the differences between the two CRISPR-Cas systems, together they may provide F. novicida with a more comprehensive defense against foreign nucleic acids. Finally, we demonstrated that the FnoCas12a and FnoCas9 machineries could be simultaneously engineered to restrict the same nonnative target, thereby expanding the toolset for prokaryotic genome manipulation. IMPORTANCE CRISPR-Cas9 and CRISPR-Cas12a systems have been widely commandeered for genome engineering. However, they originate in prokaryotes, where they function as adaptive immune systems. The details of this activity and relationship between these systems within native host organisms have been minimally explored. The human pathogen Francisella novicida contains both of these systems, with the Cas9 system also exhibiting a second activity, modulating virulence through transcriptional regulation. We compared and contrasted the ability of these two systems to control virulence and restrict DNA within their native host bacterium, highlighting differences and similarities in these two functions. Collectively, our results indicate that these two distinct and reprogrammable endogenous systems provide F. novicida with a more comprehensive defense against mobile genetic elements.

Beyond defense against foreign DNA, the CRISPR-Cas9 system of pathogenic Francisella novicida represses expression of an endogenous immunostimulatory lipoprotein and is essential for virulence. We investigated the specificity and molecular mechanism of this regulation, demonstrating that Cas9 has a highly specific regulon of four genes which must be repressed for bacterial virulence. Regulation occurs through a PAM-dependent interaction of Cas9 with its endogenous DNA targets, directed by a non-canonical small RNA (scaRNA) duplexed with tracrRNA. The limited complementarity between scaRNA and the endogenous DNA targets precludes cleavage. This highlights the evolution of the scaRNA to direct transcriptional interference via interaction with endogenous DNA without lethally targeting the chromosome. We show that scaRNA can be reprogrammed to repress other genes, and with engineered, extended complementarity to an exogenous target, the repurposed scaRNA:tracrRNA-Cas9 machinery can also be licensed to direct cleavage of target DNA. Natural Cas9 transcriptional interference likely represents a broad paradigm of regulatory functionality, which is potentially critical to the physiology of numerous Cas9-encoding pathogenic and commensal organisms.

Significance The clustered, regularly interspaced, short palindromic repeats associated endonuclease, Cas9, has quickly become a revolutionary tool in genome engineering. Utilizing small guiding RNAs, Cas9 can be targeted to specific DNA sequences of interest, where it catalyzes DNA cleavage. We now demonstrate that Cas9 from the Gram-negative bacterium Francisella novicida (FnCas9) can be reprogrammed to target a specific RNA substrate, the genome of the +ssRNA virus, hepatitis C virus, in eukaryotic cells. Further, this targeting results in inhibition of viral protein production. Overall, programmable Cas9-mediated viral RNA targeting likely represents one of myriad potential applications of FnCas9 in RNA targeting in eukaryotic cells.

The high genetic diversity of HIV-1 impedes high throughput, large-scale sequencing and full-length genome cloning by common restriction enzyme based methods. Applying novel methods that employ a high-fidelity polymerase for amplification and an unbiased fusion-based cloning strategy, we have generated several HIV-1 full-length genome infectious molecular clones from an epidemiologically linked transmission pair. These clones represent the transmitted/founder virus and phylogenetically diverse non-transmitted variants from the chronically infected individual׳s diverse quasispecies near the time of transmission. We demonstrate that, using this approach, PCR-induced mutations in full-length clones derived from their cognate single genome amplicons are rare. Furthermore, all eight non-transmitted genomes tested produced functional virus with a range of infectivities, belying the previous assumption that a majority of circulating viruses in chronic HIV-1 infection are defective. Thus, these methods provide important tools to update protocols in molecular biology that can be universally applied to the study of human viral pathogens.

Clustered, regularly interspaced, short palindromic repeats-CRISPR associated (CRISPR-Cas) systems defend bacteria against foreign nucleic acids, such as during bacteriophage infection and transformation, processes which cause envelope stress. It is unclear if these machineries enhance membrane integrity to combat this stress. Here, we show that the Cas9-dependent CRISPR-Cas system of the intracellular bacterial pathogen Francisella novicida is involved in enhancing envelope integrity through the regulation of a bacterial lipoprotein. This action ultimately provides increased resistance to numerous membrane stressors, including antibiotics. We further find that this previously unappreciated function of Cas9 is critical during infection, as it promotes evasion of the host innate immune absent in melanoma 2/apoptosis associated speck-like protein containing a CARD (AIM2/ASC) inflammasome. Interestingly, the attenuation of the cas9 mutant is complemented only in mice lacking both the AIM2/ASC inflammasome and the bacterial lipoprotein sensor Toll-like receptor 2, but not in single knockout mice, demonstrating that Cas9 is essential for evasion of both pathways. These data represent a paradigm shift in our understanding of the function of CRISPR-Cas systems as regulators of bacterial physiology and provide a framework with which to investigate the roles of these systems in myriad bacteria, including pathogens and commensals.

Purpose of review: CRISPR-Cas systems are prokaryotic immune systems against invading nucleic acids that adapt as new environmental threats arise. There are emerging examples of CRISPR-Cas functions in bacterial physiology beyond their role in adaptive immunity. This highlights the poorly understood, but potentially common, moonlighting functions of these abundant systems. We propose that these noncanonical CRISPR-Cas activities have evolved to respond to stresses at the cell envelope. Recent findings: Here, we discuss recent literature describing the impact of the extracellular environment on the regulation of CRISPR-Cas systems, and the influence of CRISPR-Cas activity on bacterial physiology. These described noncanonical CRISPR-Cas functions allow the bacterial cell to respond to the extracellular environment, primarily through changes in envelope physiology. Summary: This review discusses the expanding noncanonical functions of CRISPR-Cas systems, including their roles in virulence, focusing mainly on their relationship to the cell envelope. We first examine the effects of the extracellular environment on regulation of CRISPR-Cas components, and then discuss the impact of CRISPR-Cas systems on bacterial physiology, concentrating on their roles in influencing interactions with the environment including host organisms.

Prokaryotes use diverse strategies to improve fitness in the face of different environmental threats and stresses, including those posed by mobile genetic elements (e.g., bacteriophages and plasmids). To defend against these elements, many bacteria and archaea use elegant, RNA-directed, nucleic acid–targeting adaptive restriction machineries called CRISPR–Cas (CRISPR-associated) systems. While providing an effective defense against foreign genetic elements, these systems have also been observed to play critical roles in regulating bacterial physiology during environmental stress. Increasingly, CRISPR–Cas systems, in particular the Type II systems containing the Cas9 endonuclease, have been exploited for their ability to bind desired nucleic acid sequences, as well as direct sequence-specific cleavage of their targets. Cas9-mediated genome engineering is transcending biological research as a versatile and portable platform for manipulating genetic content in myriad systems. Here, we present a systematic overview of CRISPR–Cas history and biology, highlighting the revolutionary tools derived from these systems, which greatly expand the molecular biologists’ toolkit.