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1.
Brain Res ; 1837: 148985, 2024 May 05.
Artículo en Inglés | MEDLINE | ID: mdl-38714228

RESUMEN

OBJECTIVE: We decided to investigate the changes of global and local connectivity in anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis patients based on eigenvector centrality (EC) and regional homogeneity (ReHo). We sought new biomarkers to identify the patients based on multivariate pattern analysis (MVPA). METHODS: Functional MRI (fMRI) was performed on all participants. EC, ReHo and MVPA were used to analyze the fMRI images. The correlation between the global or local connectivity and neuropsychology tests was detected. RESULTS: The MoCA scores of the patients were lower than those of the healthy controls (HCs), while the HAMD24 and HAMA scores of the patients were higher than those of the HCs. Increased EC values in the right calcarine (CAL.R) and decreased EC values in the right putamen (PUT.R) distinguished these subjects with anti-NMDAR encephalitis from HCs. The higher ReHo values in the left postcentral gyrus (PoCG.L) were detected in the patients. The correlation analysis showed that the EC values in the PUT.R were negatively correlated with HAMD24 and HAMA scores, while the ReHo values in the PoCG.L were negatively correlated with MoCA scores. Better classification performance was reached in the EC-based classifier (AUC = 0.80), while weaker classification performance was achieved in the ReHo-based classifier (AUC = 0.74) or the classifier based on EC and ReHo (AUC = 0.74). The brain areas with large weights were located in the frontal lobe, parietal lobe, cerebellum and basal ganglia. CONCLUSION: Our findings suggest that abnormal global and local connectivity may play an important part in the pathophysiological mechanism of neuropsychiatric symptoms in the anti-NMDAR encephalitis patients. The EC-based classifier may be better than the ReHo-based classifier in identifying anti-NMDAR encephalitis patients.

2.
Analyst ; 2024 May 03.
Artículo en Inglés | MEDLINE | ID: mdl-38700251

RESUMEN

Microparticle rotation is an important process in biomedical engineering, such as biosensors, cell injection or cell morphology. Single particle rotation has been widely investigated, while rotation of particle chains has gained rare attention. In this paper, we utilize a noncontact manipulation method to rotate microparticle chains via electrorotation by designing an octuple-electrode array (OEA). Finite element simulations were conducted for analyzing the desired electrode field and optimizing the structure of microelectrode pairs. The direction of the electric field in the workspace is investigated with different voltage signal inputs through specially designed circuits. In the experiment, microparticles are driven to form several chains in the proposed electrode fields. With the rotation of the electric field, particle chains could be rotated synchronously. Automated rotation and detection of polystyrene microspheres and yeast cell chains are achieved using machine vision technology. Results show that the proposed method could be utilized to rotate ordered microparticles with an appropriate input signal.

3.
Nucleic Acids Res ; 52(2): 977-997, 2024 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-38033325

RESUMEN

Guide RNAs offer programmability for CRISPR-Cas9 genome editing but also add challenges for delivery. Chemical modification, which has been key to the success of oligonucleotide therapeutics, can enhance the stability, distribution, cellular uptake, and safety of nucleic acids. Previously, we engineered heavily and fully modified SpyCas9 crRNA and tracrRNA, which showed enhanced stability and retained activity when delivered to cultured cells in the form of the ribonucleoprotein complex. In this study, we report that a short, fully stabilized oligonucleotide (a 'protecting oligo'), which can be displaced by tracrRNA annealing, can significantly enhance the potency and stability of a heavily modified crRNA. Furthermore, protecting oligos allow various bioconjugates to be appended, thereby improving cellular uptake and biodistribution of crRNA in vivo. Finally, we achieved in vivo genome editing in adult mouse liver and central nervous system via co-delivery of unformulated, chemically modified crRNAs with protecting oligos and AAV vectors that express tracrRNA and either SpyCas9 or a base editor derivative. Our proof-of-concept establishment of AAV/crRNA co-delivery offers a route towards transient editing activity, target multiplexing, guide redosing, and vector inactivation.


Asunto(s)
Edición Génica , ARN Guía de Sistemas CRISPR-Cas , Animales , Ratones , Distribución Tisular , ARN/genética , Oligonucleótidos
4.
Small ; 20(6): e2304531, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-37789506

RESUMEN

More and more attention has been paid to lithium-sulfur (Li─S) batteries due to their high energy density and low cost. However, the intractable "shuttle effect" and the low conductivity of S and its reaction product, Li2 S, compromise battery performance. To address the inherent challenges, a hollow composite catalyst as a separator coating material is designed, in which CoFe alloy is embedded in a carbon skeleton (CoFeNC@NC). In the hybrid structure, the carbon layer can endow the batteries with high electrical conductivity, while the CoFe alloy can effectively bidirectionally catalyze the conversion between lithium polysulfides (LiPSs) and Li2 S, accelerating the reaction kinetics and reducing the dissolution of LiPSs. Furthermore, the distinctive hollow structure with a cracked surface can facilitate the exposure of a more accessible catalytically active site and enhance Li+ diffusion. Benefiting from the synergistic effects, Li─S batteries with a CoFeNC@NC catalyst achieve a high sulfur utilization (1250.8 mAh g-1 at 0.2 C), superior rate performance (756 mAh g-1 at 2 C), and excellent cycling stability (an ultralow capacity fading of 0.054% per cycle at 1 C for 1000 cycles). Even at a sulfur loading of 5.3 mg cm-2 , a high area capacity of 4.05 mAh cm-2 can still be achieved after 100 cycles, demonstrating its potential practicality.

5.
CRISPR J ; 6(6): 570-582, 2023 12.
Artículo en Inglés | MEDLINE | ID: mdl-38108517

RESUMEN

CRISPR-based genome-editing technologies, including nuclease editing, base editing, and prime editing, have recently revolutionized the development of therapeutics targeting disease-causing mutations. To advance the assessment and development of genome editing tools, a robust mouse model is valuable, particularly for evaluating in vivo activity and delivery strategies. In this study, we successfully generated a knock-in mouse line carrying the Traffic Light Reporter design known as TLR-multi-Cas variant 1 (TLR-MCV1). We comprehensively validated the functionality of this mouse model for both in vitro and in vivo nuclease and prime editing. The TLR-MCV1 reporter mouse represents a versatile and powerful tool for expediting the development of editing technologies and their therapeutic applications.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Animales , Ratones , Sistemas CRISPR-Cas/genética , Modelos Animales de Enfermedad , Endonucleasas/genética , Tecnología
6.
bioRxiv ; 2023 Oct 29.
Artículo en Inglés | MEDLINE | ID: mdl-37904991

RESUMEN

Prime editing efficiency is modest in cells that are quiescent or slowly proliferating where intracellular dNTP levels are tightly regulated. MMLV-reverse transcriptase - the prime editor polymerase subunit - requires high intracellular dNTPs levels for efficient polymerization. We report that prime editing efficiency in primary cells and in vivo is increased by mutations that enhance the enzymatic properties of MMLV-reverse transcriptase and can be further complemented by targeting SAMHD1 for degradation.

7.
Brain Res ; 1820: 148605, 2023 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-37775074

RESUMEN

OBJECTIVE: To explore potential mechanisms of cognitive changes in patients with anti-NMDAR encephalitis (ANMDARE) from intramodule and intermodule effects of brain functional networks. METHODS: Resting-state functional MRI(rs-fMRI) imaging data was collected from 30 ANMDARE and 30 healthy controls (HCs). A brain functional matrix was constructed, and sparsity was established by module similarity. For both groups, changes in functional connectivity (FC) within and between modules was calculated, and whole-brain functional topology was analyzed. Finally, the association of brain functional with cognitive function in ANMDARE was further analyzed. RESULTS: Compared to HCs, ANMDARE had enhanced connectivity within the modules that included the occipito-parietal-temporal and parahippocampal gyri. ANMDARE had significantly higher participation coefficients (PC) in the right inferior frontal gyrus than HCs and significantly lower PC in the left superior parietal lobule, left caudate nucleus, and right putamen. No statistically significant differences in global topological properties were found between the two groups. No correlations were found between functional and structural brain indicators and the Cognitive Assessment Scale and the Emotional Deficit Scale. CONCLUSIONS: Patients with ANMDARE are manifested by enhanced intramodular FC and intermodular connectivity changes in the brain. This may help to understand the pathophysiological mechanisms of the disease from a global perspective.


Asunto(s)
Encefalitis Antirreceptor N-Metil-D-Aspartato , Humanos , Encefalitis Antirreceptor N-Metil-D-Aspartato/diagnóstico por imagen , Receptores de N-Metil-D-Aspartato , Encéfalo/diagnóstico por imagen , Mapeo Encefálico , Cognición , Imagen por Resonancia Magnética/métodos
8.
Nat Biotechnol ; 2023 Sep 14.
Artículo en Inglés | MEDLINE | ID: mdl-37709915

RESUMEN

Reverse transcriptases, used in prime editing systems, exhibit lower fidelity, processivity and dNTP affinity than many DNA-dependent DNA polymerases. We report that a DNA-dependent DNA polymerase (phi29), untethered from Cas9, enables editing from a synthetic, end-stabilized DNA-containing template at up to 60% efficiency in human cells. Compared to prime editing, DNA polymerase editing avoids autoinhibitory intramolecular base pairing of the template, facilitates template synthesis and supports larger insertions (>100 nucleotides).

9.
Nat Methods ; 20(6): 898-907, 2023 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-37156841

RESUMEN

Prime editors have a broad range of potential research and clinical applications. However, methods to delineate their genome-wide editing activities have generally relied on indirect genome-wide editing assessments or the computational prediction of near-cognate sequences. Here we describe a genome-wide approach for the identification of potential prime editor off-target sites, which we call PE-tag. This method relies on the attachment or insertion of an amplification tag at sites of prime editor activity to allow their identification. PE-tag enables genome-wide profiling of off-target sites in vitro using extracted genomic DNA, in mammalian cell lines and in the adult mouse liver. PE-tag components can be delivered in a variety of formats for off-target site detection. Our studies are consistent with the high specificity previously described for prime editor systems, but we find that off-target editing rates are influenced by prime editing guide RNA design. PE-tag represents an accessible, rapid and sensitive approach for the genome-wide identification of prime editor activity and the evaluation of prime editor safety.


Asunto(s)
Edición Génica , Genoma , Ratones , Animales , Edición Génica/métodos , ADN/genética , Roturas del ADN de Doble Cadena , Línea Celular , Sistemas CRISPR-Cas , Mamíferos/genética
10.
bioRxiv ; 2023 Mar 23.
Artículo en Inglés | MEDLINE | ID: mdl-36993169

RESUMEN

Guide RNAs offer programmability for CRISPR-Cas9 genome editing but also add challenges for delivery. Chemical modification, which has been key to the success of oligonucleotide therapeutics, can enhance the stability, distribution, cellular uptake, and safety of nucleic acids. Previously, we engineered heavily and fully modified SpyCas9 crRNA and tracrRNA, which showed enhanced stability and retained activity when delivered to cultured cells in the form of the ribonucleoprotein complex. In this study, we report that a short, fully stabilized oligonucleotide (a "protecting oligo"), which can be displaced by tracrRNA annealing, can significantly enhance the potency and stability of a heavily modified crRNA. Furthermore, protecting oligos allow various bioconjugates to be appended, thereby improving cellular uptake and biodistribution of crRNA in vivo. Finally, we achieved in vivo genome editing in adult mouse liver and central nervous system via co-delivery of unformulated, chemically modified crRNAs with protecting oligos and AAV vectors that express tracrRNA and either SpyCas9 or a base editor derivative. Our proof-of-concept establishment of AAV/crRNA co-delivery offers a route towards transient editing activity, target multiplexing, guide redosing, and vector inactivation.

11.
Neurol Sci ; 44(4): 1341-1350, 2023 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-36571641

RESUMEN

BACKGROUND AND PURPOSE: Limited studies had jointly excavated the structural and functional changes in cognitive deficit in anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis patients. We aimed to explore these changes in anti-NMDAR patients and their effect on cognitive function. METHODS: Twenty-three patients and 25 healthy controls (HCs) underwent resting-state functional magnetic resonance imaging, diffusion tensor imaging scanning, and neuroethology tests. The significantly differentiated brain regions via the fractional amplitude of low-frequency fluctuation (fALFF) were defined as regions of interest (ROIs). Granger causal, functional connectivity, and tract-based spatial statistical analyses were applied to explore the functional changes in ROIs and assess the structural changes. RESULTS: HCs outperformed patients in Montreal Cognitive Assessment. The fALFF values of right gyrus rectus (RGR) in patients were significantly reduced. The fractional anisotropy (FA) values of WM in the genu of corpus callosum and right superior corona radiata were significantly decreased and positively associated with neuroethology testing scores. The Granger causal connectivity (GCC) from the left inferior parietal lobule to RGR was significantly decreased and positively associated with inherent vigilance. Indicated by the multiple linear regression result, decreased FA value of the right superior corona radiata might be a reliable marker that reflects the cognitive impairment. CONCLUSIONS: Significant changes in spontaneous neural activities, GCC, and WM structures in anti-NMDAR encephalitis were reported. These findings promote the understanding of underlying relationships between cerebral function, structural network alterations, and cognitive dysfunction.


Asunto(s)
Encefalitis Antirreceptor N-Metil-D-Aspartato , Sustancia Blanca , Humanos , Imagen de Difusión Tensora/métodos , Imagen por Resonancia Magnética/métodos , Encefalitis Antirreceptor N-Metil-D-Aspartato/diagnóstico por imagen , Sustancia Blanca/diagnóstico por imagen , Sustancia Blanca/patología , Encéfalo
12.
J Neuroimaging ; 33(1): 156-166, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36085558

RESUMEN

BACKGROUND AND PURPOSE: To investigate the longitudinal alterations of cortical structural-functional coupling (SF coupling) in patients with temporal lobe epilepsy (TLE) over a 2-year follow-up, thereby exploring the neuropathophysiological mechanisms of TLE. METHODS: Twenty-eight TLE patients and 42 age- and gender-matched healthy controls (HCs) were recruited. We used resting-state functional MRI and diffusion-weighted imaging to estimate and compare SF coupling at the multiscale network level (whole-brain, modular, and regional levels). Then, we analyzed the relationships between the spatial patterns of SF coupling, the principal functional connectivity (FC) gradient, and the functional participation coefficient (PC). Finally, we related regional SF coupling changes between baseline and follow-up to the expression of regional TLE-specific genes. RESULTS: Compared with HCs, TLE patients showed higher baseline SF couplings within the whole-brain, limbic, and default-mode modules. SF couplings within visual and dorsal attention modules were increased at follow-up compared to baseline. In all three groups, the spatial patterns of SF coupling aligned with the principal FC gradient and the functional PC. The longitudinal change in regional SF coupling in TLE patients was significantly positively correlated with the expression of the CUX2 gene. CONCLUSIONS: Aberrant SF coupling was revealed in TLE and related to macroscale cortical hierarchies, functional segregation, and TLE-specific gene expression; these data help increase our understanding of the neuropathophysiological mechanisms underlying TLE.


Asunto(s)
Epilepsia del Lóbulo Temporal , Humanos , Epilepsia del Lóbulo Temporal/diagnóstico por imagen , Imagen por Resonancia Magnética/métodos , Encéfalo , Atención , Imagen de Difusión por Resonancia Magnética
13.
Front Neurosci ; 16: 871128, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35837122

RESUMEN

Objective: To investigate the changes in the cerebellar-cerebral language network in temporal lobe epilepsy (TLE) patients from the cerebellar perspective, the research analyzes the changes of language and cognitive network in terms of functional connectivity (FC), as well as their efficiency of the reorganization were evaluated basing on relationship between the network metrics and neuropsychological scale scores. Methods: 30 TLE patients and 30 healthy controls were recruited. Brain activity was evaluated by voxel-mirrored homotopic connectivity analysis (VMHC). Two groups were analyzed and compared in terms of language FC using the following methods: Seed-to-Voxel analysis, pairwise correlations [region of interest(ROI)-to-ROI] and graph theory. Correlation analysis was performed between network properties and neuropsychological score. Results: Compared with healthy participants, VMHC values in the Cerebellum Anterior Lobe, Frontal Lobe, Frontal_Sup_R/L, Cingulum_Ant_R/L, and Cingulum_Mid_R/L were decreased in TLE patients. Decreased FC was observed from the Cerebelum_10_R to the left inferior frontal gyrus, from the Cerebelum_6_R to the left Lingual Gyrus, from the Cerebelum_4_5_R to left Lingual Gyrus, left Cuneal Cortex and Precuneous Cortex, from the Cerebelum_3_R to Brain-Stem, and from the Cerebelum_Crus1_L to Cerebelum_6_R in TLE patients. The FC was enhanced between bilateral Cingulum_Mid and angular gyrus and frontoparietal insular cranium, between Frontal_Sup_Med L and left/right superior temporal gyrus (pSTG l/r), while it was decreased between left middle temporal gyrus and pSTG l/r. Compared with controls, the Betweenness Centrality (BC) of the right superior marginal gyrus (SMG), Temporal_Pole_Mid_R and Temporal_Mid_L as well as the Degree Centrality (DC) and Nodal Efficiency (NE) of the right SMG were lower in TLE patients. Further analysis showed that decreased VMHC in bilateral Cerebellum Anterior Lobe was positively correlated with the Boston Naming Test score in TLE patients, but it was negatively correlated with the Verbal Fluency Test score. The NE and DC of SMG_R were both negatively correlated with visual perception score in Montreal Cognitive Assessment. Conclusion: Our results suggest that presence of abnormalities in the static functional connectivity and the language and cognitive network of TLE patients. Cerebellum potentially represents an intervention target for delaying or improving language and cognitive deficits in patients with TLE.

14.
J Environ Manage ; 319: 115720, 2022 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-35853308

RESUMEN

Bio-drying can significantly reduce the moisture content of waste. The factors, functional microorganisms, and the transformation of main components were investigated during bio-drying of perishable waste. This study provides a scientific basis for the improvement of the bio-drying process and the necessity for secondary pollutant control. Reaction temperature and microbial biomass were main factors during the bio-drying process. The ideal bio-drying conditions included an initial temperature above 20 °C, intermittent ventilation, and appropriate microbial inoculation. The main microorganisms included Alcaligenes, Aquamicrobium, and Brevundimonas. From each gram of the carbonaceous, nitrogenous, sulfur-containing compounds, and phosphorus-containing substances in the perishable waste, approximately 0.74 g, 0.66 g, 0.40 g, and 0.94 g, respectively, were transferred as gas-phase products; consisting mainly of ammonia and volatile organic compounds: 2-heptanone, dimethyl heptanone, and benzene. In the leachate, the respective amounts of the carbonaceous, nitrogenous, sulfur-containing compounds, and phosphorus-containing substances were 3.20 × 10-3 g, 4.08 × 10-3 g, 0.33 g, and 9.52 × 10-3 g, while those of the residual substances remaining in solid were 0.26 g, 0.33 g, 0.28 g, and 0.05 g.


Asunto(s)
Desecación , Compuestos Orgánicos Volátiles , Nitrógeno , Fósforo , Azufre , Compuestos de Azufre
15.
Front Psychiatry ; 13: 888150, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35722568

RESUMEN

Purpose: Previous research has shown that subcortical brain regions are related to vigilance in temporal lobe epilepsy (TLE). However, it is unknown whether alterations in the function and structure of basal forebrain (BF) subregions are associated with vigilance impairment in distinct kinds of TLE. We aimed to investigate changes in the structure and function BF subregions in TLE patients with and without focal to bilateral tonic-clonic seizures (FBTCS) and associated clinical features. Methods: A total of 50 TLE patients (25 without and 25 with FBTCS) and 25 healthy controls (HCs) were enrolled in this study. The structural and functional alterations of BF subregions in TLE were investigated using voxel-based morphometry (VBM) and resting-state functional connectivity (rsFC) analysis. Correlation analyses were utilized to investigate correlations between substantially altered imaging characteristics and clinical data from patients. Results: FBTCS patients had a lower rsFC between Ch1-3 and the bilateral striatum as well as the left cerebellum posterior lobe than non-FBTCS patients. In comparison to non-FBTCS patients, the rsFC between Ch4 and the bilateral amygdala was also lower in FBTCS patients. Compared to HCs, the TLE patients had reduced rsFC between the BF subregions and the cerebellum, striatum, default mode network, frontal lobe, and occipital lobes. In the FBTCS group, the rsFC between the left Ch1-3 and striatum was positive correlated with the vigilance measures. In the non-FBTCS group, the rsFC between the left Ch4 and striatum was significantly negative correlated with the alertness measure. Conclusion: These results extend current understanding of the pathophysiology of impaired vigilance in TLE and imply that the BF subregions may serve as critical nodes for developing and categorizing TLE biomarkers.

16.
Neuroradiology ; 64(10): 2021-2030, 2022 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-35618843

RESUMEN

PURPOSE: Previous studies have discovered different neuroimaging features in anti-NMDAR encephalitis associated with cognitive dysfunction. However, it is unknown whether there is a correlation between abnormal homotopic connectivity and cognitive impairment in anti-NMDAR encephalitis. We aim to explore the homotopic connectivity patterns of patients with anti-NMDAR encephalitis and their associations with clinical characteristics. METHODS: Resting-state functional magnetic resonance imaging (rs-fMRI) was performed on 29 patients with anti-NMDAR encephalitis and 26 healthy controls (HCs). Voxel-mirrored homotopic connectivity (VMHC) and multivariate pattern analysis (MVPA) were applied to analyze the imaging data. A correlation was also performed between aberrant brain regions and clinical parameters. RESULTS: Compared to HCs, the performance of alertness in the patient group was typically worse (p < 0.05). A significant decrease in VMHC was observed in many regions of the patients in comparison to HCs, including the cerebellar 6, para-hippocampal gyrus, insula, precuneus, and middle frontal gyrus (p < 0.001). The insula and middle frontal gyrus were found to show positive correlations with alertness. The MVPA method achieved a classification accuracy of 74.55% with a sensitivity of 82.76% and a specificity of 65.38% in discriminating patients from HCs. CONCLUSION: Our findings indicate that interhemispheric functional imbalance may play a significant role in the pathophysiology of cognitive dysfunction in anti-NMDAR encephalitis. The MVPA results suggest that abnormal VMHC may play a crucial role in the identification of patients with anti-NMDAR encephalitis from HCs.


Asunto(s)
Encefalitis Antirreceptor N-Metil-D-Aspartato , Disfunción Cognitiva , Encefalitis Antirreceptor N-Metil-D-Aspartato/diagnóstico por imagen , Encéfalo/diagnóstico por imagen , Humanos , Imagen por Resonancia Magnética/métodos , Neuroimagen
17.
ACS Appl Mater Interfaces ; 14(21): 24386-24395, 2022 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-35594421

RESUMEN

Aqueous zinc-ion batteries (ZIBs), especially the aqueous zinc-manganese batteries, have received considerable attention due to their low cost, safety, and environmental benignity. However, manganese oxide cathode materials usually suffer from unsatisfactory cycling stability. In this study, we report an interface engineering strategy to improve the performance of the Mn-based cathode electrode for ZIBs. Both the results of experiments and density functional theory confirmed that SnO2 can act as a "glue" to strengthen the interfacial interaction between the conductive graphene substrate and MnOOH, which plays a vital role during the charging/discharging process of manganese oxide. By this interface engineering strategy, the cycling stability of the in situ deposited Mn-based electrode was significantly improved, and a specific capacity of 271 mA h g-1 can be retained even after 1500 cycles. This study may provide a thought or establish a framework for the rational design of high-performance cathode materials for ZIBs via interface engineering.

18.
ACS Chem Biol ; 17(5): 1045-1050, 2022 05 20.
Artículo en Inglés | MEDLINE | ID: mdl-35446558

RESUMEN

CRISPR-Cas technology has revolutionized genome editing. Its broad and fast-growing application in biomedical research and therapeutics has led to increased demand for guide RNAs. The synthesis of chemically modified single-guide RNAs (sgRNAs) containing >100 nucleotides remains a bottleneck. Here we report the development of a tetrazine ligation method for the preparation of sgRNAs. A tetrazine moiety on the 3'-end of the crRNA and a norbornene moiety on the 5'-end of the tracrRNA enable successful ligation between crRNA and tracrRNA to form sgRNA under mild conditions. Tetrazine-ligated sgRNAs allow efficient genome editing of reporter and endogenous loci in human cells. High-efficiency editing requires structural optimization of the linker.


Asunto(s)
Edición Génica , ARN Guía de Kinetoplastida , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Humanos , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/genética
19.
Nat Biotechnol ; 40(9): 1388-1393, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-35379962

RESUMEN

Delivery and optimization of prime editors (PEs) have been hampered by their large size and complexity. Although split versions of genome-editing tools can reduce construct size, they require special engineering to tether the binding and catalytic domains. Here we report a split PE (sPE) in which the Cas9 nickase (nCas9) remains untethered from the reverse transcriptase (RT). The sPE showed similar efficiencies in installing precise edits as the parental unsplit PE3 and no increase in insertion-deletion (indel) byproducts. Delivery of sPE to the mouse liver with hydrodynamic injection to modify ß-catenin drove tumor formation with similar efficiency as PE3. Delivery with two adeno-associated virus (AAV) vectors corrected the disease-causing mutation in a mouse model of type I tyrosinemia. Similarly, prime editing guide RNAs (pegRNAs) can be split into a single guide RNA (sgRNA) and a circular RNA RT template to increase flexibility and stability. Compared to previous sPEs, ours lacks inteins, protein-protein affinity modules and nuclease-sensitive pegRNA extensions, which increase construct complexity and might reduce efficiency. Our modular system will facilitate the delivery and optimization of PEs.


Asunto(s)
ARN Circular , Tirosinemias , Animales , Sistemas CRISPR-Cas , Desoxirribonucleasa I/genética , Edición Génica , Ratones , ARN Circular/genética , ARN Guía de Kinetoplastida/genética , ADN Polimerasa Dirigida por ARN/genética , Tirosinemias/genética
20.
ACS Appl Mater Interfaces ; 14(6): 8282-8296, 2022 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-35112830

RESUMEN

Hierarchical, ultrathin, and porous NiMoO4@CoMoO4 on Co3O4 hollow bones were successfully designed and synthesized by a hydrothermal route from the Co-precursor, followed by a KOH (potassium hydroxide) activation process. The hydrothermally synthesized Co3O4 nanowires act as the scaffold for anchoring the NiMoO4@CoMoO4 units but also show more compatibility with NiMoO4, leading to high conductivity in the heterojunction. The intriguing morphological features endow the hierarchical Co3O4@NiMoO4@CoMoO4 better electrochemical performance where the capacity of the Co3O4@NiMoO4@CoMoO4 heterojunction being 272 mA·h·g-1 at 1 A·g-1 can be achieved with a superior retention of 84.5% over 1000 cycles. The enhanced utilization of single/few NiMoO4@CoMoO4 shell layers on the Co3O4 core make it easy to accept extra electrons, enhancing the adsorption of OH- at the shell surface, which contribute to the high capacity. In our work, an asymmetric supercapacitor utilizing the optimized Co3O4@NiMoO4@CoMoO4 activated carbon (AC) as electrode materials was assembled, namely, Co3O4@NiMoO4@CoMoO4//AC device, yielding a maximum high energy density of 53.9 W·h·kg-1 at 1000 W·kg-1. It can retain 25.92 W·h·kg-1 even at 8100 W·kg-1, revealing its potential and viability for applications. The good power densities are ascribed to the porous feature from the robust architecture with recreated abundant mesopores on the composite, which assure improved conductivity and enhanced diffusion of OH- and also the electron transport. The work demonstrated here holds great promise for synthesizing other heterojunction materials M3O4@MMoO4@MMoO4 (M = Fe, Ni, Sn, etc).

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