2′-O-Methyl modified guide RNA promotes the single nucleotide polymorphism (SNP) discrimination ability of CRISPR–Cas12a systems

The CRISPR–Cas12a system has been widely applied to genome editing and molecular diagnostics. However, off-target cleavages and false-positive results remain as major concerns in Cas12a practical applications. Herein, we propose a strategy by utilizing the 2′-O-methyl (2′-OMe) modified guide RNA (gRNA) to promote the Cas12a''s specificity. Gibbs free energy analysis demonstrates that the 2′-OMe modifications at the 3′-end of gRNA effectively suppress the Cas12a''s overall non-specific affinity while maintaining high on-target affinity. For general application illustrations, HBV genotyping and SARS-CoV-2 D614G mutant biosensing platforms are developed to validate the enhanced Cas12a''s specificity. Our results indicate that the 2′-OMe modified gRNAs could discriminate single-base mutations with at least two-fold enhanced specificity compared to unmodified gRNAs. Furthermore, we investigate the enhancing mechanisms of the 2′-OMe modified Cas12a systems by molecular docking simulations and the results suggest that the 2′-OMe modifications at the 3′-end of gRNA reduce the Cas12a''s binding activity to off-target DNA. This work offers a versatile and universal gRNA design strategy for highly specific Cas12a system development.

This study illustrates that 2′-O-methyl modified gRNAs improve the specificity of the CRISPR–Cas12a system (mg-CRISPR) via suppressing the Cas12a''s affinity to off-target DNA and provides an efficient strategy for high-specificity gRNA design.     

MnO2 nanosheets as a carrier and accelerator for improved live-cell biosensing application of CRISPR/Cas12a

Besides gene-editing, the CRISPR/Cas12a system has also been widely used in in vitro biosensing, but its applications in live-cell biosensing are rare. One reason is lacking appropriate carriers to synchronously deliver all components of the CRISPR/Cas12a system into living cells. Herein, we demonstrate that MnO₂ nanosheets are an excellent carrier of CRISPR/Cas12a due to the two important roles played by them. Through a simple mixing operation, all components of the CRISPR/Cas12a system can be loaded on MnO₂ nanosheets and thus synchronously delivered into cells. Intracellular glutathione (GSH)-induced decomposition of MnO₂ nanosheets not only results in the rapid release of the CRISPR/Cas12a system in cells but also provides Mn²⁺ as an accelerator to promote CRISPR/Cas12a-based biosensing of intracellular targets. Due to the merits of highly efficient delivery, rapid intracellular release, and the accelerated signal output reaction, MnO₂ nanosheets work better than commercial liposome carriers in live-cell biosensing analysis of survivin messenger RNA (mRNA), producing much brighter fluorescence images in a shorter time. The use of MnO₂ nanosheets might provide a good carrier for different CRISPR/Cas systems and achieve the rapid and sensitive live-cell biosensing analysis of different intracellular targets, thus paving a promising way to promote the applications of CRISPR/Cas systems in living cells.

Herein, we demonstrate that MnO₂ nanosheets are an excellent carrier of CRISPR/Cas12a due to the two important roles played by them.     

Amplified detection of nucleic acids and proteins using an isothermal proximity CRISPR Cas12a assay

Herein, we describe an isothermal proximity CRISPR Cas12a assay that harnesses the target-induced indiscrimitive single-stranded DNase activity of Cas12a for the quantitative profiling of gene expression at the mRNA level and detection of proteins with high sensitivity and specificity. The target recognition is achieved through proximity binding rather than recognition by CRISPR RNA (crRNA), which allows for flexible assay design. A binding-induced primer extension reaction is used to generate a predesigned CRISPR-targetable sequence as a barcode for further signal amplification. Through this dual amplification protocol, we were able to detect as low as 1 fM target nucleic acid and 100 fM target protein isothermally. The practical applicability of this assay was successfully demonstrated for the temporal profiling of interleukin-6 gene expression during allergen-mediated mast cell activation.

Herein, we develop an isothermal proximity CRISPR Cas12a assay that harnesses the target-induced collateral cleavage activity of Cas12a for the quantitative profiling of gene expression and detection of proteins with high sensitivity and specificity.     

Engineering CRISPR/Cas-based nanosystems for therapeutics,diagnosis and bioimaging

CRISPR/Cas system has been utilized to rationally manipulate intracellular genes, and it has been engineered as versatile and efficient gene editing tools with precise site-specificity and excellent targeting ability for therapeutics, diagnostics, and bioimaging. Here, the evolution and application of CRISPR/Cas systems were sketched chronologically. Landmark works were exemplified to illustrate the design principles of CRISPR/Cas systems. Furthermore, the delivery vectors of CRISPR/Cas system especially DNA nanomaterials-based vectors were categorized and illuminated. DNA nanomaterials are suitable for CRISPR/Cas system delivery via base pairing due to its sequence programmability and biocompatibility. Then the applications of CRISPR/Cas in diagnosis and genomic imaging were highlighted. At the end of the review, the challenges and opportunities of CRISPR/Cas systems were deeply discussed. We envision that the grant advances on CRISPR/Cas systems will promote the development of interdisciplinary fields in chemistry, biology and medicine.     

Photocontrol of CRISPR/Cas9 function by site-specific chemical modification of guide RNA

The function of CRISPR/Cas9 can be conditionally controlled by the rational engineering of guide RNA (gRNA) to target the gene of choice for precise manipulation of the genome. Particularly, chemically modified gRNA that can be activated by using specific stimuli provides a unique tool to expand the versatility of conditional control. Herein, unlike previous engineering of gRNA that generally focused on the RNA part only but neglected RNA–protein interactions, we aimed at the interactive sites between 2′-OH of ribose in the seed region of gRNA and the Cas9 protein and identified that chemical modifications at specific sites could be utilized to regulate the Cas9 activity. By introducing a photolabile group at these specific sites, we achieved optical control of Cas9 activity without disrupting the Watson–Crick base pairing. We further examined our design through CRISPR-mediated gene activation and nuclease cleavage in living cells and successfully manipulated the gene expression by using light irradiation. Our site-specific modification strategy exhibited a highly efficient and dynamic optical response and presented a new perspective for manipulating gRNA based on the RNA–protein interaction rather than the structure of RNA itself. In addition, these specific sites could also be potentially utilized for modification of other stimuli-responsive groups, which would further enrich the toolbox for conditional control of CRISPR/Cas9 function.

The CRISPR/Cas9 function is optically controlled in living cells by the site-specifically caged guide RNA based on the RNA–protein interaction.     

Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens

We report on the development of an on-chip RPA (recombinase polymerase amplification) with simultaneous multiplex isothermal amplification and detection on a solid surface. The isothermal RPA was applied to amplify specific target sequences from the pathogens Neisseria gonorrhoeae, Salmonella enterica and methicillin-resistant Staphylococcus aureus (MRSA) using genomic DNA. Additionally, a positive plasmid control was established as an internal control. The four targets were amplified simultaneously in a quadruplex reaction. The amplicon is labeled during on-chip RPA by reverse oligonucleotide primers coupled to a fluorophore. Both amplification and spatially resolved signal generation take place on immobilized forward primers bount to expoxy-silanized glass surfaces in a pump-driven hybridization chamber. The combination of microarray technology and sensitive isothermal nucleic acid amplification at 38 °C allows for a multiparameter analysis on a rather small area. The on-chip RPA was characterized in terms of reaction time, sensitivity and inhibitory conditions. A successful enzymatic reaction is completed in <20 min and results in detection limits of 10 colony-forming units for methicillin-resistant Staphylococcus aureus and Salmonella enterica and 100 colony-forming units for Neisseria gonorrhoeae. The results show this method to be useful with respect to point-of-care testing and to enable simplified and miniaturized nucleic acid-based diagnostics. Figure

The combination of multiplex isothermal nucleic acid amplification with RPA and spatially-resolved signal generation on specific immobilized oligonucleotides     

Universal and high-fidelity DNA single nucleotide polymorphism detection based on a CRISPR/Cas12a biochip

Single nucleotide polymorphisms (SNPs) are associated with many human diseases, so accurate and efficient SNP detection is of great significance for early diagnosis and clinical prognosis. This report proposes a universal and high-fidelity genotyping method in microfluidic point-of-care equipment based on the clustered regularly interspaced short palindromic repeat (CRISPR) system. Briefly, by systematically inserting the protospacer-adjacent-motif (PAM) sequence, we improved the universality of the CRISPR/Cas12a based SNP detection; by removing the complementary ssDNA and introducing an additional nucleotide mismatch, we improved the sensitivity and specificity. We preloaded the CRISPR/Cas12a reagents into the point-of-care biochip for automating the process, increasing the stability and long-term storage. This biochip enables us to rapidly and conveniently detect the genotypes within 20 min. In a practical application, the CRISPR/Cas12a biochip successfully distinguished three genotypes (homozygous wild type; the homozygous mutant type; and the heterozygous mutant type) of the CYP1A1*2 (A4889G, rs1048943), CYP2C19*2 (G681A, rs4244285), CYP2C9*3 (A1075C, rs1057910), and CYP2C19*3 (G636A, rs4986893) genes related to multiple cancers from 17 clinical blood samples. This CRISPR/Cas12a-based SNP genotyping method, being universal, accurate, and sensitive, will have broad applications in molecular diagnostics and clinical research.

A universal and high-fidelity genotyping method based on the clustered regularly interspaced short palindromic repeat (CRISPR) system was performed on the microfluidic point-of-care equipment.     

Chemical synthesis of stimuli-responsive guide RNA for conditional control of CRISPR-Cas9 gene editing

CRISPR-Cas9 promotes changes in identity or abundance of nucleic acids in live cells and is a programmable modality of broad biotechnological and therapeutic interest. To reduce off-target effects, tools for conditional control of CRISPR-Cas9 functions are under active research, such as stimuli-responsive guide RNA (gRNA). However, the types of physiologically relevant stimuli that can trigger gRNA are largely limited due to the lack of a versatile synthetic approach in chemistry to introduce diverse labile modifications into gRNA. In this work, we developed such a general method to prepare stimuli-responsive gRNA based on site-specific derivatization of 2′-O-methylribonucleotide phosphorothioate (PS-2′-OMe). We demonstrated CRISPR-Cas9-mediated gene editing in human cells triggered by oxidative stress and visible light, respectively. Our study tackles the synthetic challenge and paves the way for chemically modified RNA to play more active roles in gene therapy.

Conditional control of CRISPR-Cas9 activity by reactive oxygen species and visible light is achieved using stimuli-responsive guide RNA synthesized by a general method based on RNA 2′-O-methylribonucleotide phosphorothioate.     

A Conformational Restriction Strategy for the Control of CRISPR/Cas Gene Editing with Photoactivatable Guide RNAs**

The CRISPR/Cas system is one of the most powerful tools for gene editing. However, approaches for precise control of genome editing and regulatory events are still desirable. Here, we report the spatiotemporal and efficient control of CRISPR/Cas9- and Cas12a-mediated editing with conformationally restricted guide RNAs (gRNAs). This approach relied on only two or three pre-installed photo-labile substituents followed by an intramolecular cyclization, representing a robust synthetic method in comparison to the heavily modified linear gRNAs that often require extensive screening and time-consuming optimization. This tactic could direct the precise cleavage of the genes encoding green fluorescent protein (GFP) and the vascular endothelial growth factor A (VEGFA) protein within a predefined cutting region without notable editing leakage in live cells. We also achieved light-mediated myostatin (MSTN) gene editing in embryos, wherein a new bow-knot-type gRNA was constructed with excellent OFF/ON switch efficiency. Overall, our work provides a significant new strategy in CRISPR/Cas editing with modified circular gRNAs to precisely manipulate where and when genes are edited.     

Spatiotemporal Control of CRISPR/Cas9 Function in Cells and Zebrafish using Light-Activated Guide RNA

We developed a new method for the conditional regulation of CRISPR/Cas9 activity in mammalian cells and zebrafish embryos using photochemically activated, caged guide RNAs (gRNAs). Caged gRNAs are generated by substituting four nucleobases evenly distributed throughout the 5′-protospacer region with caged nucleobases during synthesis. Caging confers complete suppression of gRNA:dsDNA-target hybridization and rapid restoration of CRISPR/Cas9 function upon optical activation. This tool offers simplicity and complete programmability in design, high spatiotemporal specificity in cells and zebrafish embryos, excellent off-to-on switching, and stability by preserving the ability to form Cas9:gRNA ribonucleoprotein complexes. Caged gRNAs are novel tools for the conditional control of gene editing, thereby enabling the investigation of spatiotemporally complex physiological events by obtaining a better understanding of dynamic gene regulation.     

Spatiotemporal Control of CRISPR/Cas9 Function in Cells and Zebrafish using Light‐Activated Guide RNA

We developed a new method for the conditional regulation of CRISPR/Cas9 activity in mammalian cells and zebrafish embryos using photochemically activated, caged guide RNAs (gRNAs). Caged gRNAs are generated by substituting four nucleobases evenly distributed throughout the 5′‐protospacer region with caged nucleobases during synthesis. Caging confers complete suppression of gRNA:dsDNA‐target hybridization and rapid restoration of CRISPR/Cas9 function upon optical activation. This tool offers simplicity and complete programmability in design, high spatiotemporal specificity in cells and zebrafish embryos, excellent off‐to‐on switching, and stability by preserving the ability to form Cas9:gRNA ribonucleoprotein complexes. Caged gRNAs are novel tools for the conditional control of gene editing, thereby enabling the investigation of spatiotemporally complex physiological events by obtaining a better understanding of dynamic gene regulation.     

Element coding based accurate evaluation of CRISPR/Cas9 initial cleavage

As a powerful gene editing tool, the kinetic mechanism of CRISPR/Cas9 has been the focus for its further application. Initial cleavage events as the first domino followed by nuclease end trimming significantly affect the final on-target rate. Here we propose EC-CRISPR, element coding CRISPR, an accurate evaluation platform for initial cleavage that directly characterizes the cleavage efficiency and breaking sites. We benchmarked the influence of 19 single mismatch and 3 multiple mismatch positions of DNA-sgRNA on initial cleavage, as well as various reaction conditions. Results from EC-CRISPR demonstrate that the PAM-distal single mismatch is relatively acceptable compared to the proximal one. And multiple mismatches will not only affect the cleavage efficiency, but also generate more non-site #3 cleavage. Through in-depth research of kinetic behavior, we uncovered an abnormally higher non-#3 proportion at the initial stage of cleavage by using EC-CRISPR. Together, our results provided insights into cleavage efficiency and breaking sites, demonstrating that EC-CRISPR as a novel quantitative platform for initial cleavage enables accurate comparison of efficiencies and specificities among multiple CRISPR/Cas enzymes.

Initial cleavage events as the first domino of CRISPR/Cas9 kinetic behaviors. To accurately evaluate the initial cleavage of Cas9, element coding CRISPR platform-enabled direct characterization of the cleavage efficiency and cleavage sites was proposed.     

Emerging biosensing and transducing techniques for potential applications in point-of-care diagnostics

With the deepening of our understanding in life science, molecular biology, nanotechnology, optics, electrochemistry and other areas, an increasing number of biosensor design strategies have emerged in recent years, capable of providing potential practical applications for point-of-care (POC) diagnosis in various human diseases. Compared to conventional biosensors, the latest POC biosensor research aims at improving sensor precision, cost-effectiveness and time-consumption, as well as the development of versatile detection strategies to achieve multiplexed analyte detection in a single device and enable rapid diagnosis and high-throughput screening. In this review, various intriguing strategies in the recognition and transduction of POC (from 2018 to 2021) are described in light of recent advances in CRISPR technology, electrochemical biosensing, and optical- or spectra-based biosensing. From the perspective of promoting emerging bioanalytical tools into practical POC detecting and diagnostic applications, we have summarized key advances made in this field in recent years and presented our own perspectives on future POC development and challenges.

POC diagnostics are driven by the rapid advances in CRISPR, electrochemical and optical biosensors. Related emerging strategies are described and discussed from the perspective of facilitating the practical application of biosensors in POC testing.     

Highly Efficient and Rapid Detection of the Cleavage Activity of Cas9/gRNA via a Fluorescent Reporter

The RNA-guided endonuclease clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) derived from CRISPR systems is a simple and efficient genome-editing technology applied to various cell types and organisms. So far, the extensive approach to detect the cleavage activity of customized Cas9/guide RNA (gRNA) is T7 endonuclease I (T7EI) assay, which is time and labor consuming. In this study, we developed a visualized fluorescent reporter system to detect the specificity and cleavage activity of gRNA. Two gRNAs were designed to target porcine immunoglobulin M and nephrosis 1 genes. The cleavage activity was measured by using the traditional homology-directed repair (HDR)-based fluorescent reporter and the single-strand annealing (SSA)-based fluorescent reporter we established in this study. Compared with the HDR assay, the SSA-based fluorescent reporter approach was a more efficient and dependable strategy for testing the cleavage activity of Cas9/gRNA, thereby providing a universal and efficient approach for the application of CRISPR/Cas9 in generating gene-modified cells and organisms.     

Machine learning designs non-hemolytic antimicrobial peptides

Machine learning (ML) consists of the recognition of patterns from training data and offers the opportunity to exploit large structure–activity databases for drug design. In the area of peptide drugs, ML is mostly being tested to design antimicrobial peptides (AMPs), a class of biomolecules potentially useful to fight multidrug-resistant bacteria. ML models have successfully identified membrane disruptive amphiphilic AMPs, however mostly without addressing the associated toxicity to human red blood cells. Here we trained recurrent neural networks (RNN) with data from DBAASP (Database of Antimicrobial Activity and Structure of Peptides) to design short non-hemolytic AMPs. Synthesis and testing of 28 generated peptides, each at least 5 mutations away from training data, allowed us to identify eight new non-hemolytic AMPs against Pseudomonas aeruginosa, Acinetobacter baumannii, and methicillin-resistant Staphylococcus aureus (MRSA). These results show that machine learning (ML) can be used to design new non-hemolytic AMPs.

Machine learning models trained with experimental data for antimicrobial activity and hemolysis are shown to produce new non-hemolytic antimicrobial peptides active against multidrug-resistant bacteria.     

Chronocoulometric DNA biosensor based on a glassy carbon electrode modified with gold nanoparticles, poly(dopamine) and carbon nanotubes

We describe a sensitive chronocoulometric biosensor for the sequence-specific detection of DNA. It is based on a glassy carbon electrode modified with multi-walled carbon nanotubes, polydopamine, and gold nanoparticles. The ruthenium(III)hexammine complex acts as the electrochemical indicator. Electrochemical impedance spectra and scanning electron microscopy are employed to investigate the assembly of the electrode surface. The signals of the ruthenium complex electrostatically bound to the anionic phospho groups of the DNA strands are measured by chronocoulometry before and after hybridization. The difference in signal intensity is linearly related to the logarithm of the concentration of the target DNA in the range of 1.0 nM to 10 fM with a detection limit of 3.5fM (S/N?=?3) under optimal conditions. This biosensor exhibits excellent sensitivity and selectivity and has been used for an assay of complementary target DNA in human serum sample with satisfactory results.

Bacterio- and lymphocytotoxicity of silver nanocomposite with sulfated arabinogalactan

The antibacterial activity and cytotoxicity of the silver-containing drug, which is zerovalent metallic silver nanoparticles stabilized by sulfated arabinogalactan, towards human lymphocytes were evaluated. The bacteriostatic concentration towards E. coli ATCC 25922, E. coli ESBL1224, S. aureus MRSA34R, S. aureus ATCC 29213, and P. aeruginosa ATCC 27853 and hospital strains E. coli, P. aeruginosa, and P. mirabilis ranges from 3 to 50 μg mL^?1. Their bactericidal activity varies from 5 to 100 μg mL^?1, and the concentration of the nanocomposite toxic to isolated human peripheral blood lymphocytes is 5 μg m^L?1.     

Ultrasensitive impedimetric lectin based biosensor for glycoproteins containing sialic acid

We report on an ultrasensitive label-free lectin-based impedimetric biosensor for the determination of the sialylated glycoproteins fetuin and asialofetuin. A sialic acid binding agglutinin from Sambucus nigra I was covalently immobilised on a mixed self-assembled monolayer (SAM) consisting of 11-mercaptoundecanoic acid and 6-mercaptohexanol. Poly(vinyl alcohol) was used as a blocking agent. The sensor layer was characterised by atomic force microscopy, electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy. The biosensor exhibits a linear range that spans 7 orders of magnitude for both glycoproteins, with a detection limit as low as 0.33 fM for fetuin and 0.54 fM for asialofetuin. We also show, by making control experiments with oxidised asialofetuin, that the biosensor is capable of quantitatively detecting changes in the fraction of sialic acid on glycoproteins. We conclude that this work lays a solid foundation for future applications of such a biosensor in terms of the diagnosis of diseases such as chronic inflammatory rheumatoid arthritis, genetic disorders and cancer, all of which are associated with aberrant glycosylation of protein biomarkers.

Antibacterial Activity of Chinese Red Propolis against Staphylococcus aureus and MRSA

The antibacterial activity of propolis has long been of great interest, and the chemical composition of propolis is directly dependent on its source. We recently obtained a type of propolis from China with a red color. Firstly, the antibacterial properties of this unusual propolis were determined against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA). Studies on its composition identified and quantified 14 main polyphenols of Chinese red propolis extracts (RPE); quantification was carried out using liquid chromatography triple quadrupole tandem mass spectrometry (LC-QQQ-MS/MS) and RPE was found to be rich in pinobanksin, pinobanksin-3-acetate, and chrysin. In vitro investigations of its antibacterial activity revealed that its activity against S. aureus and MRSA is due to disruption of the cell wall and cell membrane, which then inhibits bacterial growth. Despite its similar antibacterial activities against S. aureus and MRSA, metabolomic analysis further revealed the effects of RPE on bacteria metabolism were different. The untargeted metabolomic results showed that a total of 7 metabolites in 12 metabolic pathways had significant changes (Fold change > 2, p < 0.05 *) after RPE treatment in S. aureus, while 11 metabolites in 9 metabolic pathways had significant changes (Fold change > 2, p < 0.05 *) after RPE treated on MRSA. Furthermore, RPE downregulated several specific genes related to bacterial biofilm formation, autolysis, cell wall synthesis, and bacterial virulence in MRSA. In conclusion, the data obtained indicate that RPE may be a promising therapeutic agent against S. aureus and MRSA.