Cyclic Tetra-Adenylate (cA4) Recognition by Csa3; Implications for an Integrated Class 1 CRISPR-Cas Immune Response in Saccharolobus solfataricus.

Csa3 family transcription factors are ancillary CRISPR-associated proteins composed of N-terminal CARF domains and C-terminal winged helix-turn-helix domains. The activity of Csa3 transcription factors is thought to be controlled by cyclic oligoadenyate (cOA) second messengers produced by type III CRISPR-Cas surveillance complexes. Here we show that Saccharolobus solfataricus Csa3a recognizes cyclic tetra-adenylate (cA4) and that Csa3a lacks self-regulating "ring nuclease" activity present in some other CARF domain proteins. The crystal structure of the Csa3a/cA4 complex was also determined and the structural and thermodynamic basis for cA4 recognition are described, as are conformational changes in Csa3a associated with cA4 binding. We also characterized the effect of cA4 on recognition of putative DNA binding sites. Csa3a binds to putative promoter sequences in a nonspecific, cooperative and cA4-independent manner, suggesting a more complex mode of transcriptional regulation. We conclude the Csa3a/cA4 interaction represents a nexus between the type I and type III CRISPR-Cas systems present in S. solfataricus, and discuss the role of the Csa3/cA4 interaction in coordinating different arms of this integrated class 1 immune system to mount a synergistic, highly orchestrated immune response.

Small Molecule Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Inhibitors: Hit to Lead Optimization of Systemic Agents.

The optimization of a new class of small molecule PCSK9 mRNA translation inhibitors is described. The potency, physicochemical properties, and off-target pharmacology associated with the hit compound (1) were improved by changes to two regions of the molecule. The last step in the synthesis of the congested amide center was enabled by three different routes. Subtle structural changes yielded significant changes in pharmacology and off-target margins. These efforts led to the identification of 7l and 7n with overall profiles suitable for in vivo evaluation. In a 14-day toxicology study, 7l demonstrated an improved safety profile vs lead 7f. We hypothesize that the improved safety profile is related to diminished binding of 7l to nontranslating ribosomes and an apparent improvement in transcript selectivity due to the lower strength of 7l stalling of off-target proteins.

Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing.

CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo.

Correction: Selective stalling of human translation through small-molecule engagement of the ribosome nascent chain.

[This corrects the article DOI: 10.1371/journal.pbio.2001882.].

Structural studies of Acidianus tailed spindle virus reveal a structural paradigm used in the assembly of spindle-shaped viruses.

The spindle-shaped virion morphology is common among archaeal viruses, where it is a defining characteristic of many viral families. However, structural heterogeneity intrinsic to spindle-shaped viruses has seriously hindered efforts to elucidate the molecular architecture of these lemon-shaped capsids. We have utilized a combination of cryo-electron microscopy and X-ray crystallography to study Acidianus tailed spindle virus (ATSV). These studies reveal the architectural principles that underlie assembly of a spindle-shaped virus. Cryo-electron tomography shows a smooth transition from the spindle-shaped capsid into the tubular-shaped tail and allows low-resolution structural modeling of individual virions. Remarkably, higher-dose 2D micrographs reveal a helical surface lattice in the spindle-shaped capsid. Consistent with this, crystallographic studies of the major capsid protein reveal a decorated four-helix bundle that packs within the crystal to form a four-start helical assembly with structural similarity to the tube-shaped tail structure of ATSV and other tailed, spindle-shaped viruses. Combined, this suggests that the spindle-shaped morphology of the ATSV capsid is formed by a multistart helical assembly with a smoothly varying radius and allows construction of a pseudoatomic model for the lemon-shaped capsid that extends into a tubular tail. The potential advantages that this novel architecture conveys to the life cycle of spindle-shaped viruses, including a role in DNA ejection, are discussed.

Selective stalling of human translation through small-molecule engagement of the ribosome nascent chain.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating the levels of plasma low-density lipoprotein cholesterol (LDL-C). Here, we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling, we demonstrate that PF-06446846 is highly selective for the inhibition of PCSK9 translation. The mechanism of action employed by PF-06446846 reveals a previously unexpected tunability of the human ribosome that allows small molecules to specifically block translation of individual transcripts.

Regulating the ribosome: a spotlight on RNA dark matter.

In this issue, Pircher et al. (2014) show that an abundant ribosome-associated 18 nt noncoding RNA (ncRNA), derived from the open reading frame of an mRNA, acts directly on the ribosome and regulates global translation levels in response to hypertonic shock.

Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic repeat (CRISPR)-associated complex for antiviral defense (CASCADE).

In response to viral infection, many prokaryotes incorporate fragments of virus-derived DNA into loci called clustered regularly interspaced short palindromic repeats (CRISPRs). The loci are then transcribed, and the processed CRISPR transcripts are used to target invading viral DNA and RNA. The Escherichia coli "CRISPR-associated complex for antiviral defense" (CASCADE) is central in targeting invading DNA. Here we report the structural and functional characterization of an archaeal CASCADE (aCASCADE) from Sulfolobus solfataricus. Tagged Csa2 (Cas7) expressed in S. solfataricus co-purifies with Cas5a-, Cas6-, Csa5-, and Cas6-processed CRISPR-RNA (crRNA). Csa2, the dominant protein in aCASCADE, forms a stable complex with Cas5a. Transmission electron microscopy reveals a helical complex of variable length, perhaps due to substoichiometric amounts of other CASCADE components. A recombinant Csa2-Cas5a complex is sufficient to bind crRNA and complementary ssDNA. The structure of Csa2 reveals a crescent-shaped structure unexpectedly composed of a modified RNA-recognition motif and two additional domains present as insertions in the RNA-recognition motif. Conserved residues indicate potential crRNA- and target DNA-binding sites, and the H160A variant shows significantly reduced affinity for crRNA. We propose a general subunit architecture for CASCADE in other bacteria and Archaea.

The structure of the CRISPR-associated protein Csa3 provides insight into the regulation of the CRISPR/Cas system.

Adaptive immune systems have recently been recognized in prokaryotic organisms where, in response to viral infection, they incorporate short fragments of invader-derived DNA into loci called clustered regularly interspaced short palindromic repeats (CRISPRs). In subsequent infections, the CRISPR loci are transcribed and processed into guide sequences for the neutralization of the invading RNA or DNA. The CRISPR-associated protein machinery (Cas) lies at the heart of this process, yet many of the molecular details of the CRISPR/Cas system remain to be elucidated. Here, we report the first structure of Csa3, a CRISPR-associated protein from Sulfolobus solfataricus (Sso1445), which reveals a dimeric two-domain protein. The N-terminal domain is a unique variation on the dinucleotide binding domain that orchestrates dimer formation. In addition, it utilizes two conserved sequence motifs [Thr-h-Gly-Phe-(Asn/Asp)-Glu-X(4)-Arg and Leu-X(2)-Gly-h-Arg] to construct a 2-fold symmetric pocket on the dimer axis. This pocket is likely to represent a regulatory ligand-binding site. The N-terminal domain is fused to a C-terminal MarR-like winged helix-turn-helix domain that is expected to be involved in DNA recognition. Overall, the unique domain architecture of Csa3 suggests a transcriptional regulator under allosteric control of the N-terminal domain. Alternatively, Csa3 may function in a larger complex, with the conserved cleft participating in protein-protein or protein-nucleic acid interactions. A similar N-terminal domain is also identified in Csx1, a second CRISPR-associated protein family of unknown function.