Engineering of a high-fidelity Cas12a nuclease variant capable of allele-specific editing.

CRISPR-Cas12a, often regarded as a precise genome editor, still requires improvements in specificity. In this study, we used a GFP-activation assay to screen 14 new Cas12a nucleases for mammalian genome editing, successfully identifying 9 active ones. Notably, these Cas12a nucleases prefer pyrimidine-rich PAMs. Among these nucleases, we extensively characterized Mb4Cas12a obtained from Moraxella bovis CCUG 2133, which recognizes a YYN PAM (Y = C or T). Our biochemical analysis demonstrates that Mb4Cas12a can cleave double-strand DNA across a wide temperature range. To improve specificity, we constructed a SWISS-MODEL of Mb4Cas12a based on the FnCas12a crystal structure and identified 8 amino acids potentially forming hydrogen bonds at the target DNA-crRNA interface. By replacing these amino acids with alanine to disrupt the hydrogen bond, we tested the influence of each mutation on Mb4Cas12a specificity. Interestingly, the F370A mutation improved specificity with minimal influence on activity. Further study showed that Mb4Cas12a-F370A is capable of discriminating single-nucleotide polymorphisms. These new Cas12a orthologs and high-fidelity variants hold substantial promise for therapeutic applications.

Phage-assisted evolution of compact Cas9 variants targeting a simple NNG PAM.

Compact Cas9 nucleases hold great promise for therapeutic applications. Although several compact Cas9 nucleases have been developed, many genomic loci still could not be edited due to a lack of protospacer adjacent motifs (PAMs). We previously developed a compact SlugCas9 recognizing an NNGG PAM. Here we demonstrate that SlugCas9 displays comparable activity to SpCas9. We developed a simple phage-assisted evolution to engineer SlugCas9 for unique PAM requirements. Interestingly, we generated a SlugCas9 variant (SlugCas9-NNG) that could recognize an NNG PAM, expanding the targeting scope. We further developed a SlugCas9-NNG-based adenine base editor and demonstrated that it could be delivered by a single adeno-associated virus to disrupt PCSK9 splice donor and splice acceptor. These genome editors greatly enhance our ability for in vivo genome editing.

In Vivo Base Editing of Scn5a Rescues Type 3 Long QT Syndrome in Mice.

BACKGROUND: Pathogenic variants in SCN5A can result in long QT syndrome type 3, a life-threatening genetic disease. Adenine base editors can convert targeted A T base pairs to G C base pairs, offering a promising tool to correct pathogenic variants. METHODS: We generated a long QT syndrome type 3 mouse model by introducing the T1307M pathogenic variant into the Scn5a gene. The adenine base editor was split into 2 smaller parts and delivered into the heart by adeno-associated virus serotype 9 (AAV9-ABEmax) to correct the T1307M pathogenic variant. RESULTS: Both homozygous and heterozygous T1307M mice showed significant QT prolongation. Carbachol administration induced Torsades de Pointes or ventricular tachycardia for homozygous T1307M mice (20%) but not for heterozygous or wild-type mice. A single intraperitoneal injection of AAV9-ABEmax at postnatal day 14 resulted in up to 99.20% Scn5a transcripts corrected in T1307M mice. Scn5a mRNA correction rate >60% eliminated QT prolongation; Scn5a mRNA correction rate <60% alleviated QT prolongation. Partial Scn5a correction resulted in cardiomyocytes heterogeneity, which did not induce severe arrhythmias. We did not detect off-target DNA or RNA editing events in ABEmax-treated mouse hearts. CONCLUSIONS: These findings show that in vivo AAV9-ABEmax editing can correct the variant Scn5a allele, effectively ameliorating arrhythmia phenotypes. Our results offer a proof of concept for the treatment of hereditary arrhythmias.

Identification of SaCas9 orthologs containing a conserved serine residue that determines simple NNGG PAM recognition.

Due to different nucleotide preferences at target sites, no single Cas9 is capable of editing all sequences. Thus, this highlights the need to establish a Cas9 repertoire covering all sequences for efficient genome editing. Cas9s with simple protospacer adjacent motif (PAM) requirements are particularly attractive to allow for a wide range of genome editing, but identification of such Cas9s from thousands of Cas9s in the public database is a challenge. We previously identified PAMs for 16 SaCas9 orthologs. Here, we compared the PAM-interacting (PI) domains in these orthologs and found that the serine residue corresponding to SaCas9 N986 was associated with the simple NNGG PAM requirement. Based on this discovery, we identified five additional SaCas9 orthologs that recognize the NNGG PAM. We further identified three amino acids that determined the NNGG PAM requirement of SaCas9. Finally, we engineered Sha2Cas9 and SpeCas9 to generate high-fidelity versions of Cas9s. Importantly, these natural and engineered Cas9s displayed high activities and distinct nucleotide preferences. Our study offers a new perspective to identify SaCas9 orthologs with NNGG PAM requirements, expanding the Cas9 repertoire.

Ultraspecific One-Pot CRISPR-Based "Green-Yellow-Red" Multiplex Detection Strategy Integrated with Portable Cartridge for Point-of-Care Diagnosis.

Versatile, informative, sensitive, and specific nucleic acid detection plays a crucial role in point-of-care pathogen testing, genotyping, and disease monitoring. In this study, we present a novel one-pot Cas12b-based method coupled with the "Green-Yellow-Red" strategy for multiplex detection. By integrating RT-LAMP amplification and Cas12b cleavage in a single tube, the entire detection process can be completed within 1 h. Our proposed method exhibits high specificity, enabling the discrimination of single-base mutations with detection sensitivity approaching single molecule levels. Additionally, the fluorescent results can be directly observed by the naked eye or automatically analyzed using our custom-designed software Result Analyzer. To realize point-of-care detection, we developed a portable cartridge capable of both heating and fluorescence excitation. In a clinical evaluation involving 20 potentially SARS-CoV-2-infected samples, our method achieved a 100% positive detection rate when compared to standard RT-PCR. Furthermore, the identification of SARS-CoV-2 variants using our method yielded results that were consistent with the sequencing results. Notably, our proposed method demonstrates excellent transferability, allowing for the simultaneous detection of various pathogens and the identification of mutations as low as 0.5% amidst a high background interference. These findings highlight the tremendous potential of our developed method for molecular diagnostics.

Quantitative proteomics based on TMT revealed the response of PK15 cells infected PEDV wild strain.

Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), is an acute and highly contagious enteric disease with a high mortality rate in suckling piglets. Identification of proteins associated with PEDV infection may provide insights into the pathogenesis of this viral disease. In this study, we employed tandem mass tag (TMT) quantitative protein analysis to investigate proteomic changes in PK15 cells following PEDV infection, and differential protein expression profiles were obtained at 0 h, 24 h, and 48 h post-infection. Overall, a total of 6330 proteins were identified. Applying criteria for fold change >1.5 < 0.67 and p-values <0.05 resulted in the identification of 59 up-regulated proteins and 103 down-regulated proteins that exhibited significant alterations in the H24 group compared to the H0 group. The H48 group demonstrated significant upregulation of 110 proteins and downregulation of 144 proteins compared to the H0 group; additionally, there were also 10 upregulated and 30 downregulated proteins in the H48 group when compared to the H24 group. These differentially expressed proteins (DEPs) were involved in immune response regulation, signal transduction, lipid transport and metabolism processes as well as cell apoptosis pathways. Based on these DEPs, we propose that PEDV may disrupt signal transduction pathways along with lipid transport and metabolism processes leading to maximal viral replication, it may also trigger inflammatory cascades accordingly. These findings could provide valuable information for elucidating specific pathogenesis related to PEDV infection while contributing towards developing new antiviral strategies.

Genome editing with natural and engineered CjCas9 orthologs.

CjCas9 is one of the smallest CRISPR-associated (Cas9) nucleases for mammalian genome editing. However, it requires a long N4RYAC (R = A or G; Y = C or T) protospacer-adjacent motif (PAM), limiting its DNA targeting scope. In this study, we investigated the PAMs of three CjCas9 orthologs, including Hsp1Cas9, Hsp2Cas9, and CcuCas9, by performing a GFP-activation assay. Interestingly, Hsp1Cas9 and CcuCas9 recognized unique N4RAA and N4CNA PAMs, respectively. We further generated an Hsp1Cas9-Hsp2Cas9 chimeric Cas9 (Hsp1-Hsp2Cas9), which recognized a simple N4CY PAM. Genome-wide off-target analysis revealed that Hsp1-Hsp2Cas9 has very few off-targets compared to SpCas9. By analyzing the crystal structure of CjCas9, we identified eight mutations that can improve the specificity and generate a high-fidelity Hsp1-Hsp2Cas9-Y. Hsp1-Hsp2Cas9-Y enables the knockout of B4GALNT2 and CMAH in porcine fetal fibroblasts (PFFs). Moreover, we developed a high-fidelity Hsp1-Hsp2Cas9-KY which displayed undetectable off-targets revealed by GUIDE-seq at four tested loci. These natural and engineered Cas9 nucleases enabled efficient genome editing in multiple mammalian cells, expanding the DNA targeting scope.

Acute Ammonia Causes Pathogenic Dysbiosis of Shrimp Gut Biofilms.

Acute ammonia exposure has detrimental effects on shrimp, but the underlying mechanisms remain to be fully explored. In the present study, we investigated the impact of acute ammonia exposure on the gut microbiota of the white shrimp Litopenaeus vannamei and its association with shrimp mortality. Exposure to a lethal concentration of ammonia for 48 h resulted in increased mortality in L. vannamei, with severe damage to the hepatopancreas. Ammonia exposure led to a significant decrease in gut microbial diversity, along with the loss of beneficial bacterial taxa and the proliferation of pathogenic Vibrio strains. A phenotypic analysis revealed a transition from the dominance of aerobic to facultative anaerobic strains due to ammonia exposure. A functional analysis revealed that ammonia exposure led to an enrichment of genes related to biofilm formation, host colonization, and virulence pathogenicity. A species-level analysis and experiments suggest the key role of a Vibrio harveyi strain in causing shrimp disease and specificity under distinct environments. These findings provide new information on the mechanism of shrimp disease under environmental changes.

Anion-Coordination Foldamer-Based Polymer Network: from Molecular Spring to Elastomer.

Foldamer is a scaled-down version of coil spring, which can absorb and release energy by conformational change. Here, polymer networks with high density of molecular springs were developed by employing anion-coordination-based foldamers as the monomer. The coiling of the foldamer is controlled by oligo(urea) ligands coordinating to chloride ions; subsequently, the folding and unfolding of foldamer conformations endow the polymer network with excellent energy dissipation and toughness. The mechanical performance of the corresponding polymer networks shows a dramatic increase from P-L2UCl (non-folding), to P-L4UCl (a full turn), and then to P-L6UCl (1.5 turns), in terms of strength (2.62 MPa; 14.26 MPa; 22.93 MPa), elongation at break (70 %; 325 %; 352 %), Young's modulus (2.69 MPa; 63.61 MPa; 141.50 MPa), and toughness (1.12 MJ/m3; 21.39 MJ/m3; 49.62 MJ/m3), respectively, which is also better than those without anion centers and the non-foldamer based counterparts. Moreover, P-L6UCl shows enhanced strength and toughness than most of the molecular-spring based polymer networks. Thus, an effective strategy for designing high-performance anion-coordination-based materials is presented.

Transcriptomic analysis reveals impact of gE/gI/TK deletions on host response to PRV infection.

BACKGROUND: Pseudorabies virus (PRV) causes substantial losses in the swine industry worldwide. Attenuated PRV strains with deletions of immunomodulatory genes glycoprotein E (gE), glycoprotein I (gI) and thymidine kinase (TK) are candidate vaccines. However, the effects of gE/gI/TK deletions on PRV-host interactions are not well understood. METHODS: To characterize the impact of gE/gI/TK deletions on host cells, we analyzed and compared the transcriptomes of PK15 cells infected with wild-type PRV (SD2017), PRV with gE/gI/TK deletions (SD2017gE/gI/TK) using RNA-sequencing. RESULTS: The attenuated SD2017gE/gI/TK strain showed increased expression of inflammatory cytokines and pathways related to immunity compared to wild-type PRV. Cell cycle regulation and metabolic pathways were also perturbed. CONCLUSIONS: Deletion of immunomodulatory genes altered PRV interactions with host cells and immune responses. This study provides insights into PRV vaccine design.

A compact Cas9 ortholog from Staphylococcus Auricularis (SauriCas9) expands the DNA targeting scope.

Compact CRISPR/Cas9 systems that can be packaged into an adeno-associated virus (AAV) hold great promise for gene therapy. Unfortunately, currently available small Cas9 nucleases either display low activity or require a long protospacer adjacent motif (PAM) sequence, limiting their extensive applications. Here, we screened a panel of Cas9 nucleases and identified a small Cas9 ortholog from Staphylococcus auricularis (SauriCas9), which recognizes a simple NNGG PAM, displays high activity for genome editing, and is compact enough to be packaged into an AAV for genome editing. Moreover, the conversion of adenine and cytosine bases can be achieved by fusing SauriCas9 to the cytidine and adenine deaminase. Therefore, SauriCas9 holds great potential for both basic research and clinical applications.

Efficient Correction of a Hypertrophic Cardiomyopathy Mutation by ABEmax-NG.

[Figure: see text].

Discovery and engineering of small SlugCas9 with broad targeting range and high specificity and activity.

The compact CRISPR/Cas9 system, which can be delivered with their gRNA and a full-length promoter for expression by a single adeno-associated virus (AAV), is a promising platform for therapeutic applications. We previously identified a compact SauriCas9 that displays high activity and requires a simple NNGG PAM, but the specificity is moderate. Here, we identified three compact Cas9 orthologs, Staphylococcus lugdunensis Cas9 (SlugCas9), Staphylococcus lutrae Cas9 (SlutrCas9) and Staphylococcus haemolyticus Cas9 (ShaCas9), for mammalian genome editing. Of these three Cas9 orthologs, SlugCas9 recognizes a simple NNGG PAM and displays comparable activity to SaCas9. Importantly, we generated a SlugCas9-SaCas9 chimeric nuclease, which has both high specificity and high activity. We finally engineered SlugCas9 with mutations to generate a high-fidelity variant that maintains high specificity without compromising on-target editing efficiency. Our study offers important minimal Cas9 tools that are ideal for both basic research and clinical applications.

Preparation of Porous Ti/RuO2-IrO2@Pt, Ti/RuO2-TiO2@Pt and Ti/Y2O3-RuO2-TiO2@Pt Anodes for Efficient Electrocatalytic Decomposition of Tetracycline.

Electrocatalytic oxidation (ECO) has attracted attention because of its high efficiency and environmental friendliness in water treatment. The preparation of anodes with high catalytic activity and long service lifetimes is a core part of electrocatalytic oxidation technology. Here, porous Ti/RuO2-IrO2@Pt, Ti/RuO2-TiO2@Pt, and Ti/Y2O3-RuO2-TiO2@Pt anodes were fabricated by means of modified micro-emulsion and vacuum impregnation methods with high porosity titanium plates as substrates. The scanning electron microscopy (SEM) images showed that RuO2-IrO2@Pt, RuO2-TiO2@Pt, and Y2O3-RuO2-TiO2@Pt nanoparticles were coated on the inner surface of the as-prepared anodes to form the active layer. Electrochemical analysis revealed that the high porosity substrate could result in a large electrochemically active area, and a long service life (60 h at 2 A cm-2 current density, 1 mol L-1 H2SO4 as the electrolyte, and 40 °C). The degradation experiments conducted on tetracycline hydrochloride (TC) showed that the porous Ti/Y2O3-RuO2-TiO2@Pt had the highest degradation efficiency for tetracycline, reaching 100% removal in 10 min with the lowest energy consumption of 167 kWh kg-1 TOC. The reaction was consistent with the pseudo-primary kinetics results with a k value of 0.5480 mol L-1 s-1, which was 16 times higher than that of the commercial Ti/RuO2-IrO2 electrode. The fluorospectrophotometry studies verified that the degradation and mineralization of tetracycline were mainly ascribed to the •OH generated in the electrocatalytic oxidation process. This study thus presents a series of alternative anodes for future industrial wastewater treatment.

hPSC gene editing for cardiac disease therapy.

Cardiovascular diseases (CVDs) are the leading cause of mortality worldwide. However, the lack of human cardiomyocytes with proper genetic backgrounds limits the study of disease mechanisms. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have significantly advanced the study of these conditions. Moreover, hPSC-CMs made it easy to study CVDs using genome-editing techniques. This article discusses the applications of these techniques in hPSC for studying CVDs. Recently, several genome-editing systems have been used to modify hPSCs, including zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9). We focused on the recent advancement of genome editing in hPSCs, which dramatically improved the efficiency of the cell-based mechanism study and therapy for cardiac diseases.

Mechanically Interlocked Vitrimers.

Mechanically interlocked networks (MINs) have emerged as an encouraging platform for the development of mechanically robust yet adaptive materials. However, the difficulty in reversibly breaking the mechanical bonds poses a real challenge to MINs as customizable and sustainable materials. Herein, we couple the vitrimer chemistry with mechanically interlocked structures to generate a new class of MINs─referred to as mechanically interlocked vitrimers (MIVs)─to address the challenge. Specifically, we have prepared the acetoacetate-decorated [2]rotaxane that undergoes catalyst-free condensation reaction with two commercially available multiamine monomers to furnish MIVs. Compared with the control whose wheels are nonslidable under applied force, our MIVs with slidable mechanically interlocked motifs showcase enhanced mechanical performance including Young's modulus (18.5 ± 0.9 vs 1.0 ± 0.1 MPa), toughness (3.7 ± 0.1 vs 0.9 ± 0.1 MJ/m3), and damping capacity (98% vs 72%). The structural basis behind unique property profiles is demonstrated to be the force-induced host-guest dissociation and consequential intramolecular sliding of the wheels along the axles. The peculiar behaviors represent a consecutive energy dissipation mechanism, which provides a complement to other pathways that mainly depend on the breaking of sacrificial bonds. Moreover, by virtue of the vitrimer chemistry of vinylogous urethanes, we impart reprocessability and chemical recyclability to the MINs, thereby empowering the reconfiguration of the networks without breaking of the mechanical bonds. Finally, it is disclosed that the intramolecular motions of [2]rotaxanes could accelerate the dynamic exchange of the vinylogous urethane bonds via loosening the network, suggestive of a synergistic effect between the dual dynamic entities.

Mechanically Interlocked Aerogels with Densely Rotaxanated Backbones.

Mechanically interlocked molecules are considered promising candidates for the construction of self-adaptive materials by virtue of their fascinating structural and dynamic features. However, it is still a great challenge to fabricate such materials with higher complexity and richer functionality. Herein, we propose the concept of mechanically interlocked aerogels (MIAs) in which the three-dimensional (3D) porous frameworks are made of dense mechanically interlocked modules, thereby enabling the integration of mechanical adaptivity and multifunctionality in a single entity. The lightweight MIA monoliths possess a good appearance and hierarchical meso- and submicron-pores. Profiting from the combination of dynamic mechanical bonds and porous skeletons of aerogels, our MIAs are not only mechanically robust (average Young's modulus = 5.80 GPa and specific modulus = 130.5 kN·m/kg) but also showcase favorable mechanical adaptivity and responsiveness under external stimuli. Taking advantage of the above integrative merits, we demonstrate the multifunctionality of our MIAs in terms of iodine uptake, thermal insulation, and selective adsorption of organic dyes. Our work opens the door to designing intelligent aerogels with delicate topological chemical structures while facilitating the development of mechanically interlocked materials.

A chimeric PCV rescued virus with the immunogenic cap gene of PCV3 cloned into the genomic backbone of the nonpathogenic PCV1 induces specific antibodies but with no pathogenic in pigs.

A chimeric PCV called PCV1-3 with the immunogenic Cap gene of pathogenic PCV type 3(PCV3) cloned into the genomic skeleton of the nonpathogenic PCV1 was rescued and inoculated into PCV3 negative piglets. The results of fluorescence quantitative PCR showed that the PCV1-3 DNA detected in serum and tissues was negative. The pathogenicity of piglets showed that PCV 1-3 did not cause the clinical characteristics and pathological changes. The viral neutralization assay revealed that infected pigs could produce antibodies and neutralize the viral activity. All results showed that chimeric virus induced specific antibodies but with no pathogenic in pigs, which provided new candidate strains for the development of PCV3 vaccine.

Altered brain structural and functional connectivity in schizotypy.

BACKGROUND: Schizotypy refers to schizophrenia-like traits below the clinical threshold in the general population. The pathological development of schizophrenia has been postulated to evolve from the initial coexistence of 'brain disconnection' and 'brain connectivity compensation' to 'brain connectivity decompensation'. METHODS: In this study, we examined the brain connectivity changes associated with schizotypy by combining brain white matter structural connectivity, static and dynamic functional connectivity analysis of diffusion tensor imaging data and resting-state functional magnetic resonance imaging data. A total of 87 participants with a high level of schizotypal traits and 122 control participants completed the experiment. Group differences in whole-brain white matter structural connectivity probability, static mean functional connectivity strength, dynamic functional connectivity variability and stability among 264 brain sub-regions of interests were investigated. RESULTS: We found that individuals with high schizotypy exhibited increased structural connectivity probability within the task control network and within the default mode network; increased variability and decreased stability of functional connectivity within the default mode network and between the auditory network and the subcortical network; and decreased static mean functional connectivity strength mainly associated with the sensorimotor network, the default mode network and the task control network. CONCLUSIONS: These findings highlight the specific changes in brain connectivity associated with schizotypy and indicate that both decompensatory and compensatory changes in structural connectivity within the default mode network and the task control network in the context of whole-brain functional disconnection may be an important neurobiological correlate in individuals with high schizotypy.

Co-occurrence of schizo-obsessive traits and its correlation with altered executive control network functional connectivity.

The prevalence of obsessive-compulsive symptoms (OCS) in schizophrenia patients is as around 30%. Evidence suggested that mild OCS could reduce symptoms of schizophrenia, supporting the presence of compensatory functions. However, severe OCS could aggravate various impairments in schizophrenia patients, supporting the "double jeopardy hypothesis". Patients with schizo-obsessive comorbidity, schizophrenia patients and obsessive-compulsive disorder patients have been found to have similarities in executive dysfunctions and altered resting-state functional connectivity within the executive control network (ECN). Executive functions could be associated with the ECN. However, little is known as to whether such overlap exists in the subclinical populations of individuals with schizo-obsessive traits (SOT), schizotypal individuals and individuals with high levels of obsessive-compulsive symptoms (OCS). In this study, we recruited 30 schizotypal individuals, 25 individuals with OCS, 29 individuals with SOT and 29 controls for a resting-state ECN-related functional connectivity (rsFC) and a go/shift/no-go task. We found that individuals with SOT exhibited increased rsFC within the ECN compared with controls, while schizotypal individuals exhibited the opposite. Individuals with OCS exhibited decreased rsFC within the ECN and between the ECN and the default mode network (DMN), relative to controls. No significant correlational results between altered rsFC related to the ECN with executive function performance were found after corrections for multiple comparisons in three subclinical groups. Our findings showed that individuals with SOT had increased rsFC within the ECN, while schizotypal individuals and individuals with OCS showed the opposite. Our findings provide evidence for possible neural substrates of subclinical comorbidity of OCS and schizotypy.