The diversity, modularity, and efficacy of CRISPR-Cas systems are driving a biotechnological revolution. RNA-guided Cas enzymes have been adopted as tools to manipulate the genomes of cultured cells, ...animals, and plants, accelerating the pace of fundamental research and enabling clinical and agricultural breakthroughs. We describe the basic mechanisms that set the CRISPR-Cas toolkit apart from other programmable gene-editing technologies, highlighting the diverse and naturally evolved systems now functionalized as biotechnologies. We discuss the rapidly evolving landscape of CRISPR-Cas applications, from gene editing to transcriptional regulation, imaging, and diagnostics. Continuing functional dissection and an expanding landscape of applications position CRISPR-Cas tools at the cutting edge of nucleic acid manipulation that is rewriting biology.
Nuclear proteins are often given a concise title that captures their function, such as 'transcription factor,' 'polymerase' or 'nuclear-receptor.' However, for members of the Drosophila ...behavior/human splicing (DBHS) protein family, no such clean-cut title exists. DBHS proteins are frequently identified engaging in almost every step of gene regulation, including but not limited to, transcriptional regulation, RNA processing and transport, and DNA repair. Herein, we present a coherent picture of DBHS proteins, integrating recent structural insights on dimerization, nucleic acid binding modalities and oligomerization propensity with biological function. The emerging paradigm describes a family of dynamic proteins mediating a wide range of protein-protein and protein-nucleic acid interactions, on the whole acting as a multipurpose molecular scaffold. Overall, significant steps toward appreciating the role of DBHS proteins have been made, but we are only beginning to understand the complexity and broader importance of this family in cellular biology.
CRISPR adaptive immunity pathways protect prokaryotic cells against foreign nucleic acids using CRISPR RNA (crRNA)-guided nucleases. In type VI-A CRISPR-Cas systems, the signature protein Cas13a ...(formerly C2c2) contains two separate ribonuclease activities that catalyze crRNA maturation and ssRNA degradation. The Cas13a protein family occurs across different bacterial phyla and varies widely in both protein sequence and corresponding crRNA sequence conservation. Although grouped phylogenetically together, we show that the Cas13a enzyme family comprises two distinct functional groups that recognize orthogonal sets of crRNAs and possess different ssRNA cleavage specificities. These functional distinctions could not be bioinformatically predicted, suggesting more subtle co-evolution of Cas13a enzymes. Additionally, we find that Cas13a pre-crRNA processing is not essential for ssRNA cleavage, although it enhances ssRNA targeting for crRNAs encoded internally within the CRISPR array. We define two Cas13a protein subfamilies that can operate in parallel for RNA detection both in bacteria and for diagnostic applications.
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•Cas13a family contains two distinct subfamilies•Cas13a subfamilies possess distinct nuclease substrate preferences•Cas13a subfamilies also use orthogonal crRNAs•crRNA processing is not essential for ssRNA targeting by Cas13a
East-Seletsky et al. demonstrate that the CRISPR-Cas type VI-A protein family contains surprising functional variability. They define two enzyme subfamilies that use orthogonal guide RNAs and cleave with distinct substrate preferences.
Detection of viruses by innate immune sensors induces protective antiviral immunity. The viral DNA sensor cyclic GMP-AMP synthase (cGAS) is necessary for detection of HIV by human dendritic cells and ...macrophages. However, synthesis of HIV DNA during infection is not sufficient for immune activation. The capsid protein, which associates with viral DNA, has a pivotal role in enabling cGAS-mediated immune activation. We now find that NONO is an essential sensor of the HIV capsid in the nucleus. NONO protein directly binds capsid with higher affinity for weakly pathogenic HIV-2 than highly pathogenic HIV-1. Upon infection, NONO is essential for cGAS activation by HIV and cGAS association with HIV DNA in the nucleus. NONO recognizes a conserved region in HIV capsid with limited tolerance for escape mutations. Detection of nuclear viral capsid by NONO to promote DNA sensing by cGAS reveals an innate strategy to achieve distinction of viruses from self in the nucleus.
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•HIV-2, not HIV-1, activates innate immunity in macrophages and dendritic cells•NONO protein binds to the HIV-2 capsid protein with more affinity than HIV-1•NONO is an innate immune sensor of the HIV capsid in the nucleus•NONO associates with the sensor cGAS in the nucleus and enables sensing of HIV DNA
The cellular factor NONO activates cGAS-mediated innate immune defenses against HIV-2 infection via viral capsid binding.
DNA capture by a CRISPR-Cas9-guided adenine base editor Lapinaite, Audrone; Knott, Gavin J; Palumbo, Cody M ...
Science (American Association for the Advancement of Science),
07/2020, Letnik:
369, Številka:
6503
Journal Article
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CRISPR-Cas-guided base editors convert A•T to G•C, or C•G to T•A, in cellular DNA for precision genome editing. To understand the molecular basis for DNA adenosine deamination by adenine base editors ...(ABEs), we determined a 3.2-angstrom resolution cryo-electron microscopy structure of ABE8e in a substrate-bound state in which the deaminase domain engages DNA exposed within the CRISPR-Cas9 R-loop complex. Kinetic and structural data suggest that ABE8e catalyzes DNA deamination up to ~1100-fold faster than earlier ABEs because of mutations that stabilize DNA substrates in a constrained, transfer RNA-like conformation. Furthermore, ABE8e's accelerated DNA deamination suggests a previously unobserved transient DNA melting that may occur during double-stranded DNA surveillance by CRISPR-Cas9. These results explain ABE8e-mediated base-editing outcomes and inform the future design of base editors.
Structures of the CRISPR genome integration complex Wright, Addison V.; Liu, Jun-Jie; Knott, Gavin J. ...
Science (American Association for the Advancement of Science),
09/2017, Letnik:
357, Številka:
6356
Journal Article
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CRISPR-Cas systems depend on the Cas1-Cas2 integrase to capture and integrate short foreign DNA fragments into the CRISPR locus, enabling adaptation to new viruses. We present crystal structures of ...Cas1-Cas2 bound to both donor and target DNA in intermediate and product integration complexes, as well as a cryo–electron microscopy structure of the full CRISPR locus integration complex, including the accessory protein IHF (integration host factor). The structures show unexpectedly that indirect sequence recognition dictates integration site selection by favoring deformation of the repeat and the flanking sequences. IHF binding bends the DNA sharply, bringing an upstream recognition motif into contact with Cas1 to increase both the specificity and efficiency of integration. These results explain how the Cas1-Cas2 CRISPR integrase recognizes a sequence-dependent DNA structure to ensure site-selective CRISPR array expansion during the initial step of bacterial adaptive immunity.
CRISPR-Cas13a enzymes are RNA-guided, RNA-activated RNases. Their properties have been exploited as powerful tools for RNA detection, RNA imaging, and RNA regulation. However, the relationship ...between target RNA binding and HEPN (higher eukaryotes and prokaryotes nucleotide binding) domain nuclease activation is poorly understood. Using sequencing experiments coupled with in vitro biochemistry, we find that Cas13a target RNA binding affinity and HEPN-nuclease activity are differentially affected by the number and the position of mismatches between the guide and the target. We identify a central binding seed for which perfect base pairing is required for target binding and a separate nuclease switch for which imperfect base pairing results in tight binding, but not HEPN-nuclease activation. These results demonstrate that the binding and cleavage activities of Cas13a are decoupled, highlighting a complex specificity landscape. Our findings underscore a need to consider the range of effects off-target recognition has on Cas13a RNA binding and cleavage behavior for RNA-targeting tool development.
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•Cas13a RNA binding and RNase activity are differentially affected by mismatches•Perfect base pairing in the guide seed region is required for stable RNA binding•RNA mismatches in a different guide region result in binding, but not RNase activity•The RNA binding and RNase activities of Cas13a can be decoupled
CRISPR-Cas13a is a RNA-guided RNase that has emerged as a powerful tool for programmable RNA recognition. Tambe et al. find that Lbu-Cas13a target RNA binding affinity and RNase activity are differentially affected by mismatches between the guide and the target RNA and that these activities can be decoupled from each other.
Prion-like domains (PLDs) are low complexity sequences found in RNA binding proteins associated with the neurodegenerative disorder amyotrophic lateral sclerosis. Recently, PLDs have been implicated ...in mediating gene regulation via liquid-phase transitions that drive ribonucleoprotein granule assembly. In this paper, we report many PLDs in proteins associated with paraspeckles, subnuclear bodies that form around long noncoding RNA. We mapped the interactome network of paraspeckle proteins, finding enrichment of PLDs. We show that one protein, RBM14, connects key paraspeckle subcomplexes via interactions mediated by its PLD. We further show that the RBM14 PLD, as well as the PLD of another essential paraspeckle protein, FUS, is required to rescue paraspeckle formation in cells in which their endogenous counterpart has been knocked down. Similar to FUS, the RBM14 PLD also forms hydrogels with amyloid-like properties. These results suggest a role for PLD-mediated liquid-phase transitions in paraspeckle formation, highlighting this nuclear body as an excellent model system for understanding the perturbation of such processes in neurodegeneration.
The identification of pathways necessary for photoreceptor and retinal pigment epithelium (RPE) function is critical to uncover therapies for blindness. Here we report the discovery of adiponectin ...receptor 1 (AdipoR1) as a regulator of these cells' functions. Docosahexaenoic acid (DHA) is avidly retained in photoreceptors, while mechanisms controlling DHA uptake and retention are unknown. Thus, we demonstrate that AdipoR1 ablation results in DHA reduction. In situ hybridization reveals photoreceptor and RPE cell AdipoR1 expression, blunted in AdipoR1(-/-) mice. We also find decreased photoreceptor-specific phosphatidylcholine containing very long-chain polyunsaturated fatty acids and severely attenuated electroretinograms. These changes precede progressive photoreceptor degeneration in AdipoR1(-/-) mice. RPE-rich eyecup cultures from AdipoR1(-/-) reveal impaired DHA uptake. AdipoR1 overexpression in RPE cells enhances DHA uptake, whereas AdipoR1 silencing has the opposite effect. These results establish AdipoR1 as a regulatory switch of DHA uptake, retention, conservation and elongation in photoreceptors and RPE, thus preserving photoreceptor cell integrity.
This study compares the effectiveness and representativeness of electrofishing, snorkelling, seining, baited lift netting, multi‐mesh gillnetting, baited fish traps, fyke netting, angling and ...longline fishing, considering three typical lentic flood‐plain habitats at different times of day. Electrofishing was by far the most effective method yielding highest species richness, species trait representation and catch per unit of effort (CPUE), followed by seining. For single species like dace Leuciscus leuciscus, European ruffe Gymnocephalus cernua, common bream Abramis brama and silver bream Blicca bjoerkna, seining was more effective than electrofishing. With both methods, some species were more consistently caught during night, dusk or dawn than during daylight. All other methods tested cannot be generally recommended for fish community assessments in shallow backwaters due to their low representativeness of species inventory and generally low CPUE. Based on these results, electrofishing of 30 m transect replicates at different times of day for monitoring the fish community in shallow backwaters, can be recommended, enabling the maximum possible comparability to adjacent river habitats. Seining should be considered as an alternative if accessibility of habitats is restricted or electrofishing is prohibited. The 25 species detected in the backwaters also suggest that these habitats contribute a large proportion of fish diversity and should be included in standard assessments of river ecological status.
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