An adenine base editor variant expands context compatibility.

Adenine base editors (ABEs) are precise gene-editing agents that convert A:T pairs into G:C through a deoxyinosine intermediate. Existing ABEs function most effectively when the target A is in a TA context. Here we evolve the Escherichia coli transfer RNA-specific adenosine deaminase (TadA) to generate TadA8r, which extends potent deoxyadenosine deamination to RA (R = A or G) and is faster in processing GA than TadA8.20 and TadA8e, the two most active TadA variants reported so far. ABE8r, comprising TadA8r and a Streptococcus pyogenes Cas9 nickase, expands the editing window at the protospacer adjacent motif-distal end and outperforms ABE7.10, ABE8.20 and ABE8e in correcting disease-associated G:C-to-A:T transitions in the human genome, with a controlled off-target profile. We show ABE8r-mediated editing of clinically relevant sites that are poorly accessed by existing editors, including sites in PCSK9, whose disruption reduces low-density lipoprotein cholesterol, and ABCA4-p.Gly1961Glu, the most frequent mutation in Stargardt disease.

Profiling of RNA-binding protein binding sites by in situ reverse transcription-based sequencing.

RNA-binding proteins (RBPs) regulate diverse cellular processes by dynamically interacting with RNA targets. However, effective methods to capture both stable and transient interactions between RBPs and their RNA targets are still lacking, especially when the interaction is dynamic or samples are limited. Here we present an assay of reverse transcription-based RBP binding site sequencing (ARTR-seq), which relies on in situ reverse transcription of RBP-bound RNAs guided by antibodies to identify RBP binding sites. ARTR-seq avoids ultraviolet crosslinking and immunoprecipitation, allowing for efficient and specific identification of RBP binding sites from as few as 20 cells or a tissue section. Taking advantage of rapid formaldehyde fixation, ARTR-seq enables capturing the dynamic RNA binding by RBPs over a short period of time, as demonstrated by the profiling of dynamic RNA binding of G3BP1 during stress granule assembly on a timescale as short as 10 minutes.

An engineered hypercompact CRISPR-Cas12f system with boosted gene-editing activity.

Compact CRISPR-Cas systems offer versatile treatment options for genetic disorders, but their application is often limited by modest gene-editing activity. Here we present enAsCas12f, an engineered RNA-guided DNA endonuclease up to 11.3-fold more potent than its parent protein, AsCas12f, and one-third of the size of SpCas9. enAsCas12f shows higher DNA cleavage activity than wild-type AsCas12f in vitro and functions broadly in human cells, delivering up to 69.8% insertions and deletions at user-specified genomic loci. Minimal off-target editing is observed with enAsCas12f, suggesting that boosted on-target activity does not impair genome-wide specificity. We determine the cryo-electron microscopy (cryo-EM) structure of the AsCas12f-sgRNA-DNA complex at a resolution of 2.9 Å, which reveals dimerization-mediated substrate recognition and cleavage. Structure-guided single guide RNA (sgRNA) engineering leads to sgRNA-v2, which is 33% shorter than the full-length sgRNA, but with on par activity. Together, the engineered hypercompact AsCas12f system enables robust and faithful gene editing in mammalian cells.

An outbreak of Amanita exitialis poisoning.

BACKGROUND: The mushroom Amanita exitialis is reported to cause acute liver injury. It is found in Southern China, and has been previously associated with a high incidence of mortality. METHODS: We described a series of 10 patients with Amanita exitialis poisoning admitted to The Second Affiliated Hospital of the Chinese University of Hong Kong (Shenzhen) in April 2022. Patient demographics, clinical features, laboratory results, therapeutic interventions, and outcome data were collected. RESULTS: Among the 10 patients, 9 survived, while 1 died. Gastrointestinal symptoms were the first to appear (average latency period, 11 ± 4.2 h). Diarrhea was the most common clinical symptom (average duration, 4.4 days). Abdominal distention was an important sign, especially in severely-ill patients. Thrombocytopenia occurred on day 2 after mushroom ingestion and persisted for 3-4 days. Alanine aminotransferase and total bilirubin peaked on days 2-3. CONCLUSION: Amanita exitialis poisoning is characterized by gastrointestinal symptoms and liver injury. In the patient who died, acute hepatic failure led to hepatic encephalopathy and cerebral edema. Abdominal distension accompanied by thrombocytopenia was common in critically ill patients in this outbreak.

Transcriptome-wide profiling and quantification of N6-methyladenosine by enzyme-assisted adenosine deamination.

N6-methyladenosine (m6A), the most abundant internal messenger RNA modification in higher eukaryotes, serves myriad roles in regulating cellular processes. Functional dissection of m6A is, however, hampered in part by the lack of high-resolution and quantitative detection methods. Here we present evolved TadA-assisted N6-methyladenosine sequencing (eTAM-seq), an enzyme-assisted sequencing technology that detects and quantifies m6A by global adenosine deamination. With eTAM-seq, we analyze the transcriptome-wide distribution of m6A in HeLa and mouse embryonic stem cells. The enzymatic deamination route employed by eTAM-seq preserves RNA integrity, facilitating m6A detection from limited input samples. In addition to transcriptome-wide m6A profiling, we demonstrate site-specific, deep-sequencing-free m6A quantification with as few as ten cells, an input demand orders of magnitude lower than existing quantitative profiling methods. We envision that eTAM-seq will enable researchers to not only survey the m6A landscape at unprecedented resolution, but also detect m6A at user-specified loci with a simple workflow.

Experimental and Kinetic Studies of Ethylene Glycol Autoignition at High Temperatures.

As one of the simplest polyols with chemical properties of alcohol, ethylene glycol is considered as a renewable energy source and a model fuel for pyrolysis oil. In this work, autoignition characteristics of ethylene glycol have been investigated behind reflected shock waves. Experiments were conducted at pressures of 2, 5, and 10 atm, equivalence ratios of 0.5, 1.0, and 2.0, and temperatures ranging from approximately 1200 to 1600 K. The fuel concentration was also varied. Results show that the ignition delay time increases with decreasing the pressure or fuel concentration. A strong positive dependence upon the equivalence ratio was found. A quantitative relationship has been yielded by the regression analysis of the experimental data. Simulations were carried out using chemical kinetic mechanisms available in the literature to assess the reliability of mechanism. Reaction pathway and sensitivity analysis confirmed the importance of H-abstraction reactions in ethylene glycol oxidation process. Finally, a comparison between ethylene glycol and ethanol ignition was conducted. Ethylene glycol ignites faster than ethanol because of the early accumulation of H and OH radicals in the oxidation of ethylene glycol.

Structure-Activity Relationships of the Enterococcal Cytolysin.

Enterococcal cytolysin is a hemolytic virulence factor linked to human disease and increased patient mortality. Produced by pathogenic strains of Enterococcus faecalis, cytolysin is made up of two small, post-translationally modified peptides called CylLL" and CylLS". They exhibit a unique toxicity profile where lytic activity is observed for both mammalian cells and Gram-positive bacteria that is dependent on the presence of both peptides. In this study, we performed alanine substitution of all residues in CylLL" and CylLS" and determined the effect on both activities. We identified key residues involved in overall activity and residues that dictate cell type specificity. All (methyl)lanthionines as well as a Gly-rich hinge region were critical for both activities. In addition, we investigated the binding of the two subunits to bacterial cells suggesting that the large subunit CylLL" has stronger affinity for the membrane or a target molecule therein. Genome mining identified other potential two-component lanthipeptides and provided insights into potential evolutionary origins.

Structural determinants of macrocyclization in substrate-controlled lanthipeptide biosynthetic pathways.

Lanthipeptides are characterized by thioether crosslinks formed by post-translational modifications. The cyclization process that favors a single ring pattern over many other possible ring patterns has been the topic of much speculation. Recent studies suggest that for some systems the cyclization pattern and stereochemistry is determined not by the enzyme, but by the sequence of the precursor peptide. However, the factors that govern the outcome of the cyclization process are not understood. This study presents the three-dimensional structures of seven lanthipeptides determined by nuclear magnetic resonance spectroscopy, including five prochlorosins and the two peptides that make up cytolysin, a virulence factor produced by Enterococcus faecalis that is directly linked to human disease. These peptides were chosen because their substrate sequence determines either the ring pattern (prochlorosins) or the stereochemistry of cyclization (cytolysins). We present the structures of prochlorosins 1.1, 2.1, 2.8, 2.10 and 2.11, the first three-dimensional structures of prochlorosins. Our findings provide insights into the molecular determinants of cyclization as well as why some prochlorosins may be better starting points for library generation than others. The structures of the large and small subunits of the enterococcal cytolysin show that these peptides have long helical stretches, a rare observation for lanthipeptides characterized to date. These helices may explain their pore forming activity and suggest that the small subunit may recognize a molecular target followed by recruitment of the large subunit to span the membrane.

Correction: Structural determinants of macrocyclization in substrate-controlled lanthipeptide biosynthetic pathways.

[This corrects the article DOI: 10.1039/D0SC01651A.].

CylA is a sequence-specific protease involved in toxin biosynthesis.

CylA is a subtilisin-like protein belonging to a recently expanded serine protease family related to class II lanthipeptide biosynthesis. As a leader peptidase, CylA is responsible for maturation of the enterococcal cytolysin, a lantibiotic important for Enterococcus faecalis virulence. In vitro reconstitution of CylA reveals that it accepts both linear and modified cytolysin peptides with a preference for cyclized peptides. Further characterization indicates that CylA activates itself by removing its N-terminal 95 amino acids. CylA achieves sequence-specific traceless cleavage of non-cognate peptides even if they are post-translationally modified, which makes the peptidase a powerful tool for mining novel lanthipeptides by providing a general strategy for leader peptide removal. Knowledge about the substrate specificity of CylA may also facilitate the development of protease inhibitors targeting cytolysin biosynthesis as a potential therapeutic approach for enterococcal infections.

Evolved Cas9 variants with broad PAM compatibility and high DNA specificity.

A key limitation of the use of the CRISPR-Cas9 system for genome editing and other applications is the requirement that a protospacer adjacent motif (PAM) be present at the target site. For the most commonly used Cas9 from Streptococcus pyogenes (SpCas9), the required PAM sequence is NGG. No natural or engineered Cas9 variants that have been shown to function efficiently in mammalian cells offer a PAM less restrictive than NGG. Here we use phage-assisted continuous evolution to evolve an expanded PAM SpCas9 variant (xCas9) that can recognize a broad range of PAM sequences including NG, GAA and GAT. The PAM compatibility of xCas9 is the broadest reported, to our knowledge, among Cas9 proteins that are active in mammalian cells, and supports applications in human cells including targeted transcriptional activation, nuclease-mediated gene disruption, and cytidine and adenine base editing. Notably, despite its broadened PAM compatibility, xCas9 has much greater DNA specificity than SpCas9, with substantially lower genome-wide off-target activity at all NGG target sites tested, as well as minimal off-target activity when targeting genomic sites with non-NGG PAMs. These findings expand the DNA targeting scope of CRISPR systems and establish that there is no necessary trade-off between Cas9 editing efficiency, PAM compatibility and DNA specificity.

Rewritable multi-event analog recording in bacterial and mammalian cells.

We present two CRISPR-mediated analog multi-event recording apparatus (CAMERA) systems that use base editors and Cas9 nucleases to record cellular events in bacteria and mammalian cells. The devices record signal amplitude or duration as changes in the ratio of mutually exclusive DNA sequences (CAMERA 1) or as single-base modifications (CAMERA 2). We achieved recording of multiple stimuli in bacteria or mammalian cells, including exposure to antibiotics, nutrients, viruses, light, and changes in Wnt signaling. When recording to multicopy plasmids, reliable readout requires as few as 10 to 100 cells. The order of stimuli can be recorded through an overlapping guide RNA design, and memories can be erased and re-recorded over multiple cycles. CAMERA systems serve as "cell data recorders" that write a history of endogenous or exogenous signaling events into permanent DNA sequence modifications in living cells.

Aptazyme-embedded guide RNAs enable ligand-responsive genome editing and transcriptional activation.

Programmable sequence-specific genome editing agents such as CRISPR-Cas9 have greatly advanced our ability to manipulate the human genome. Although canonical forms of genome-editing agents and programmable transcriptional regulators are constitutively active, precise temporal and spatial control over genome editing and transcriptional regulation activities would enable the more selective and potentially safer use of these powerful technologies. Here, by incorporating ligand-responsive self-cleaving catalytic RNAs (aptazymes) into guide RNAs, we developed a set of aptazyme-embedded guide RNAs that enable small molecule-controlled nuclease-mediated genome editing and small molecule-controlled base editing, as well as small molecule-dependent transcriptional activation in mammalian cells.

The Enterococcal Cytolysin Synthetase Coevolves with Substrate for Stereoselective Lanthionine Synthesis.

Stereochemical control is critical in natural product biosynthesis. For ribosomally synthesized and post-translationally modified peptides (RiPPs), the mechanism(s) by which stereoselectivity is achieved is still poorly understood. In this work, we focused on the stereoselective lanthionine synthesis in lanthipeptides, a major class of RiPPs formed by the addition of Cys residues to dehydroalanine (Dha) or dehydrobutyrine (Dhb). Nonenzymatic cyclization of the small subunit of a virulence lanthipeptide, the enterococcal cytolysin, resulted in the native modified peptide as the major product, suggesting that both regioselectivity and stereoselectivity are inherent to the dehydrated peptide sequence. These results support previous computational studies that a Dhx-Dhx-Xxx-Xxx-Cys motif (Dhx = Dha or Dhb; Xxx = any amino acid except Dha, Dhb, and Cys) preferentially cyclizes by attack on the Re face of Dha or Dhb. Characterization of the stereochemistry of the products formed enzymatically with substrate mutants revealed that the lanthionine synthetase actively reinforces Re face attack. These findings support the hypothesis of substrate-controlled selectivity in lanthionine synthesis but also reveal likely coevolution of substrates and lanthionine synthetases to ensure the stereoselective synthesis of lanthipeptides with defined biological activities.

The enterococcal cytolysin synthetase has an unanticipated lipid kinase fold.

The enterococcal cytolysin is a virulence factor consisting of two post-translationally modified peptides that synergistically kill human immune cells. Both peptides are made by CylM, a member of the LanM lanthipeptide synthetases. CylM catalyzes seven dehydrations of Ser and Thr residues and three cyclization reactions during the biosynthesis of the cytolysin large subunit. We present here the 2.2 Å resolution structure of CylM, the first structural information on a LanM. Unexpectedly, the structure reveals that the dehydratase domain of CylM resembles the catalytic core of eukaryotic lipid kinases, despite the absence of clear sequence homology. The kinase and phosphate elimination active sites that affect net dehydration are immediately adjacent to each other. Characterization of mutants provided insights into the mechanism of the dehydration process. The structure is also of interest because of the interactions of human homologs of lanthipeptide cyclases with kinases such as mammalian target of rapamycin.

Substrate control in stereoselective lanthionine biosynthesis.

Enzymes are typically highly stereoselective catalysts that enforce a reactive conformation on their native substrates. We report here a rare example in which the substrate controls the stereoselectivity of an enzyme-catalysed Michael-type addition during the biosynthesis of lanthipeptides. These natural products contain thioether crosslinks formed by a cysteine attack on dehydrated Ser and Thr residues. We demonstrate that several lanthionine synthetases catalyse highly selective anti-additions in which the substrate (and not the enzyme) determines whether the addition occurs from the re or si face. A single point mutation in the peptide substrate completely inverted the stereochemical outcome of the enzymatic modification. Quantum mechanical calculations reproduced the experimentally observed selectivity and suggest that conformational restraints imposed by the amino-acid sequence on the transition states determine the face selectivity of the Michael-type cyclization.

Applications of the class II lanthipeptide protease LicP for sequence-specific, traceless peptide bond cleavage.

The final step of lanthipeptide biosynthesis involves the removal of leader peptides by dedicated proteases. In vitro characterization of LicP, a class II LanP protease involved in the biosynthesis of the lantibiotic lichenicidin, revealed a self-cleavage step that removes 100 amino acids from the N-terminus. The 2.35 Å resolution crystal structure provides insights into the active site geometry and substrate specificity, and unveiled an unusual calcium-independent maturation mechanism of a subtilisin family member. LicP processes LicA2 peptides with or without post-translational modifications, but dehydrated and cyclized LicA2 is favored. Investigation of its substrate specificity demonstrated that LicP can serve as an efficient sequence-specific traceless protease and may have great utility in basic research and biotechnology. Encouraged by these findings for LicP, we identified 13 other class II LanPs, ten of which were previously unknown, and suggest that these proteins may serve as a pool of proteases with diverse recognition sequences for general traceless tag removal applications, expanding the current toolbox of proteases.

Enrichment of the ß-catenin-TCF complex at the S and G2 phases ensures cell survival and cell cycle progression.

Wnt-ß-catenin (ß-catenin is also known as CTNNB1 in human) signaling through the ß-catenin-TCF complex plays crucial roles in tissue homeostasis. Wnt-stimulated ß-catenin-TCF complex accumulation in the nucleus regulates cell survival, proliferation and differentiation through the transcription of target genes. Compared with their levels in G1, activation of the receptor LRP6 and cytosolic ß-catenin are both upregulated in G2 cells. However, accumulation of the Wnt pathway negative regulator AXIN2 also occurs in this phase. Therefore, it is unclear whether Wnt signaling is active in G2 phase cells. Here, we established a bimolecular fluorescence complementation (BiFC) biosensor system for the direct visualization of the ß-catenin-TCF interaction in living cells. Using the BiFC biosensor and co-immunoprecipitation experiments, we demonstrate that levels of the nucleus-localized ß-catenin-TCF complex increase during the S and G2 phases, and declines in the next G1 phase. Accordingly, a subset of Wnt target genes is transcribed by the ß-catenin-TCF complex during both the S and G2 phases. By contrast, transient inhibition of this complex disturbs both cell survival and G2/M progression. Our results suggest that in S and G2 phase cells, Wnt-ß-catenin signaling is highly active and functions to ensure cell survival and cell cycle progression.