The CRISPR/Cas9 system has emerged as an important tool in biomedical research for a wide range of applications, with significant potential for genome engineering and gene therapy. In order to achieve conditional control of the CRISPR/Cas9 system, a genetically encoded light-activated Cas9 was engineered through the site-specific installation of a caged lysine amino acid. Several potential lysine residues were identified as viable caging sites that can be modified to optically control Cas9 function, as demonstrated through optical activation and deactivation of both exogenous and endogenous gene function.

The Gram-positive obligate anaerobe Clostridium difficile causes potentially fatal intestinal diseases. How this organism regulates virulence gene expression is poorly understood. In many bacterial species, the second messenger cyclic di-GMP (c-di-GMP) negatively regulates flagellar motility and, in some cases, virulence. c-di-GMP was previously shown to repress motility of C. difficile. Recent evidence indicates that flagellar gene expression is tightly linked with expression of the genes encoding the two C. difficile toxins TcdA and TcdB, which are key virulence factors for this pathogen. Here, the effect of c-di-GMP on expression of the toxin genes tcdA and tcdB was determined, and the mechanism connecting flagellar and toxin gene expressions was examined. In C. difficile, increasing c-di-GMP levels reduced the expression levels of tcdA and tcdB, as well as that of tcdR, which encodes an alternative sigma factor that activates tcdA and tcdB expression. We hypothesized that the C. difficile orthologue of the flagellar alternative sigma factor SigD (FliA; 28 ) mediates regulation of toxin gene expression in response to c-di-GMP. Indeed, ectopic expression of sigD in C. difficile resulted in increased expression levels of tcdR, tcdA, and tcdB. Furthermore, sigD expression enhanced toxin production and increased the cytopathic effect of C. difficile on cultured fibroblasts. Finally, evidence is provided that SigD directly activates tcdR expression and that SigD cannot activate tcdA or tcdB expression independent of TcdR. Taken together, these data suggest that SigD positively regulates toxin genes in C. difficile and that c-di-GMP can inhibit both motility and toxin production via SigD, making this signaling molecule a key virulence gene regulator in C. difficile.

Recent advances in pseudouridine detection reveal a complex pseudouridine landscape that includes messenger RNA and diverse classes of noncoding RNA in human cells. The known molecular functions of pseudouridine, which include stabilizing RNA conformations and destabilizing interactions with varied RNA-binding proteins, suggest that RNA pseudouridylation could have widespread effects on RNA metabolism and gene expression. Here, we emphasize how much remains to be learned about the RNA targets of human pseudouridine synthases, their basis for recognizing distinct RNA sequences, and the mechanisms responsible for regulated RNA pseudouridylation. We also examine the roles of noncoding RNA pseudouridylation in splicing and translation and point out the potential effects of mRNA pseudouridylation on protein production, including in the context of therapeutic mRNAs. Expected final online publication date for the Annual Review of Genetics, Volume 54 is November 23, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Circular RNAs (circRNAs) are long-lived, covalently closed RNAs that are abundantly expressed and evolutionarily conserved across eukaryotes. Possible functions ranging from microRNA (miRNA) and RNA binding protein sponges to regulators of transcription and translation have been proposed. Here we describe the design and characterization of recombinant adeno-associated viral (AAV) vectors packaging transgene cassettes containing intronic sequences that promote backsplicing to generate circularized RNA transcripts. Using a split GFP transgene, we demonstrate the capacity of vectors containing different flanking intronic sequences to efficiently drive persistent circRNA formation in vitro. Further, translation from circRNA is efficiently driven by an internal ribosomal entry site (IRES). Upon injecting AAV vectors encoding circRNA in mice, we observed robust transgene expression in the heart, but low transduction in the liver for the intronic elements tested. Expression in the murine brain was restricted to astrocytes following systemic or intracranial administration, while intravitreal injection in the eye yielded robust transgene expression across multiple retinal cell layers. These results highlight the potential for exploiting AAV-based circRNA expression to study circRNA function and tissue-specific regulation in animal models, as well as development of therapeutic platforms using this approach.

The bacterial CRISPR endoribonuclease Csy4 has recently been described as a potential RNA processing tool. Csy4 recognizes substrate RNA through a specific 28-nt hairpin sequence and cleaves at the 3 ′ end of the stem. To further explore applicability in mammalian cells, we introduced this hairpin at various locations in mRNAs derived from reporter transgenes and systematically evaluated the effects of Csy4-mediated processing on transgene expression. Placing the hairpin in the 5 ′ UTR or immediately after the start codon resulted in efficient degradation of target mRNA by Csy4 and knockdown of transgene expression by 20-to 40-fold. When the hairpin was incorporated in the 3 ′ UTR prior to the poly(A) signal, the mRNA was cleaved, but only a modest decrease in transgene expression (∼2.5-fold) was observed. In the absence of a poly(A) tail, Csy4 rescued the target mRNA substrate from degradation, resulting in protein expression, which suggests that the cleaved mRNA was successfully translated. In contrast, neither catalytically inactive (H29A) nor binding-deficient (R115A/R119A) Csy4 mutants were able to exert any of the effects described above. Generation of a similar 3 ′ end by RNase P-mediated cleavage was unable to rescue transgene expression independent of Csy4. These results support the idea that the selective generation of the Csy4/hairpin complex resulting from cleavage of target mRNA might serve as a functional poly(A)/poly(A) binding protein (PABP) surrogate, stabilizing the mRNA and supporting translation. Although the exact mechanism(s) remain to be determined, our studies expand the potential utility of CRISPR nucleases as tools for controlling mRNA stability and translation.

Circular RNAs (circRNAs) are highly stable, covalently closed RNAs that are regulated in a spatiotemporal manner and whose functions are largely unknown. These molecules have the potential to be incorporated into engineered systems with broad technological implications. Here we describe a switch for inducing back-splicing of an engineered circRNA that relies on the CRISPR endoribonuclease, Csy4, as an activator of circularization. The endoribonuclease activity and 3' end-stabilizing properties of Csy4 are particularly suited for this task. Coexpression of Csy4 and the circRNA switch allows for the removal of downstream competitive splice sites and stabilization of the 5' cleavage product. This subsequently results in back-splicing of the 5' cleavage product into a circRNA that can translate a reporter protein from an internal ribosomal entry site (IRES). Our platform outlines a straightforward approach toward regulating splicing and could find potential applications in synthetic biology as well as in studying the properties of different circRNAs.

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