45 results about "DNA Modification" patented technology

The technology described herein relates to novel CRISPR / Cas systems and components thereof, vectors, proteins, fusion proteins, ribonucleoprotein complexes and methods of using the novel systems and components. The new system can be used in CRIS PRi applications and can be fused with any number of effector domain patterns, such as nuclease, base editor, leader editor, and the like, to conduct a variety of DNA modifications.

In one aspect, the disclosure relates to a gene modification tool that fuses a Cas protein with a polymerase. This DSB-independent approach utilizes a DNA-RNA chimeric prime-editor-like guide RNAs (chimeric pegRNAs or cpegRNAs) to enable programmable DNA sequence replacement or excision at endogenous human genomic sites. The Cas9 and RNA portion of cpegRNA helps with targeting a gene, while the DNA portion of cpegRNA is used as a template by the polymerase to repair. Furthermore, the disclosed method can be used to introduce large DNA modifications in the genome.

The present disclosure provides systems, compositions, and methods for nucleic acid modification. More particularly, the present disclosure provides systems comprising a TnpA protein, a TnpB protein, an IscB protein, or a combination thereof, and methods using thereof.

Provided herein are compositions and methods for the multiplexed profiling of RNA and DNA modifications across transcriptomes and genomes, respectively. The methods combine molecular recognition of non-canonical features (e.g., base modifications, backbone modifications, lesions, and / or structural elements) of a target nucleic acid with a step of writing the information from this recognition event into the neighboring genetic sequence of the target nucleic acid using a barcode. The resultant barcoded nucleic acids are then converted into sequencing libraries and read by DNA / RNA sequencing methods. This step reveals the sequence of the barcode, which is correlated with the non-canonical feature in the target nucleic acid(s). The high throughput profiling methods described herein allow for localization of one or more modifications in a target nucleic acid. The methods also allow for identification of the nature and location of several or all DNA / RNA modifications in parallel.

Systems and methods for producing multiple unique edits in T1 seeds of a plant are provided. In one example, the method includes transforming at least one expression cassette into a plant cell or plant tissue. The at least one expression cassette may include a nucleic acid encoding a DNA modification enzyme; optionally, a nucleic acid encoding at least one guide RNA (gRNA); and a floral mosaic (FMOS) regulatory sequence, wherein the FMOS regulatory sequence (i) mediates the expression of the DNA modification enzyme in at least one of a flower primordium cell and a flower reproductive organ, and (ii) mediates multiple edits in at least one of the flower primordium and the flower reproductive organ. The method may also include regenerating the plant cell or plant tissue into a TO plant having multiple T1 seeds, wherein the T1 seeds contain multiple unique edits.

Aspects of the present disclosure are directed to methods, compositions, and kits for detection and analysis of DNA and RNA cytosine methylation and / or RNA pseudouridylation. Certain aspects include methods, compositions and kits useful in bisulfite sequencing of methylated and / or pseudouridinylated nucleic acids, including methylated and / or pseudouridinylated nucleic acids from low-input samples such as cell-free DNA (cfDNA) and cell-free RNA, long fragment polynucleotides, and / or cfDNA and DNA from formalin-fixed paraffin-embedded (FFPE) samples. Also disclosed herein are methods, compositions and kits useful in bisulfite sequencing of methylated and / or pseudouridinylated nucleic acids with low nucleic acid damage, higher true positive results, and / or lower false negative results.

This invention relates to the field of biotechnology, and more particularly to a high-density oligoA-tailed DNA-gold nanomaterial and its preparation method. The oligoA-tailed DNA-gold nanomaterial comprises AuNPs, and / or AuNRs, and modified oligoA-tailed DNA; the oligoA-tailed DNA sequence is shown in SEQ ID No. 1. This preparation method is low-cost, easy to operate, and can achieve high-density oligoA-tailed DNA modification on the surface of gold nanoparticles with different morphologies, while exhibiting high stability.

Aspects of the present disclosure are directed to methods, compositions, and kits for detection and analysis of DNA and RNA cytosine methylation and / or RNA pseudouridylation. Certain aspects include methods, compositions and kits useful in bisulfite sequencing of methylated and / or pseudouridinylated nucleic acids, including methylated and / or pseudouridinylated nucleic acids from low-input samples such as cell-free DNA (cfDNA) and cell-free RNA, long fragment polynucleotides, and / or cfDNA and DNA from formalin-fixed paraffin-embedded (FFPE) samples. Also disclosed herein are methods, compositions and kits useful in bisulfite sequencing of methylated and / or pseudouridinylated nucleic acids with low nucleic acid damage, higher true positive results, and / or lower false negative results.

Provided herein are compositions and methods for the multiplexed profiling of RNA and DNA modifications across transcriptomes and genomes, respectively. The methods combine molecular recognition of non-canonical features (e.g., base modifications, backbone modifications, lesions, and / or structural elements) of a target nucleic acid with a step of writing the information from this recognition event into the neighboring genetic sequence of the target nucleic acid using a barcode. The resultant barcoded nucleic acids are then converted into sequencing libraries and read by DNA / RNA sequencing methods. This step reveals the sequence of the barcode, which is correlated with the non-canonical feature in the target nucleic acid(s). The high throughput profiling methods described herein allow for identification and / or localization of one or more modifications in a target nucleic acid. The methods also allow for identification of the nature and location of several or all DNA / RNA modifications in parallel.

This invention relates to a method for preparing a functional DNA gel and its applications. Addressing the problems of existing methods for immobilizing functional nucleic acids requiring DNA modification, which increases immobilization costs, and the reduction of functionality due to chemical fixation, this invention mixes functional circular DNA with a gel monomer solution, and adds an initiator and a coagulant to initiate a cross-linking polymerization reaction. During the reaction, the gel monomers pass through the functional circular DNA, polymerizing into long chains and then cross-linking into a network structure. This allows the functional circular DNA to be enriched on the network structure in a suspended, fixed form, resulting in a functional DNA gel. The functional circular DNA is circular DNA containing functional nucleic acid sequences. This invention immobilizes functional circular DNA on the gel network structure without chemical modification, and the preparation method is simple. It can be used for the efficient and specific removal of pollutants such as heavy metal ions, toxins, veterinary drugs, and pesticides from solutions, as well as the enrichment of small molecule nucleic acids in solutions.

The invention belongs to the technical field of DNA coding molecules, and provides a synthesis method of an on-DNA carbonyl compound, which comprises: providing an on-DNA amino compound and an on-DNA hydroxyl compound, the on-DNA amino compound comprising a DNA fragment and an amino group coupled to the DNA fragment, and the on-DNA hydroxyl compound comprising a DNA fragment and a hydroxyl group coupled to the DNA fragment; and reacting the on-DNA amino compound and / or the on-DNA hydroxyl compound with laccase and an auxiliary catalyst to obtain the on-DNA carbonyl compound, namely the on-DNA carbonyl compound containing a DNA fragment and carbonyl coupled with the DNA fragment. The synthesis method has the advantages of mild conditions, environmental friendliness and small damage to DNA, and is suitable for construction of a DNA coding molecular library, fluorescence labeled molecule coupling performed by using an on-DNA carbonyl reaction handle, DNA modification coupling and functional modification in targeted drug delivery.

The present disclosure provides a DNA-targeting RNA that comprises a targeting sequence and, together with a modifying polypeptide, provides for site-specific modification of a target DNA and / or a polypeptide associated with the target DNA. The present disclosure further provides site-specific modifying polypeptides. The present disclosure further provides methods of site-specific modification of a target DNA and / or a polypeptide associated with the target DNA The present disclosure provides methods of modulating transcription of a target nucleic acid in a target cell, generally involving contacting the target nucleic acid with an enzymatically inactive Cas9 polypeptide and a DNA-targeting RNA. Kits and compositions for carrying out the methods are also provided. The present disclosure provides genetically modified cells that produce Cas9; and Cas9 transgenic non-human multicellular organisms.

This disclosure to the methods for nucleic acid modification, gene targeting, and gene tagging comprising an engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated transposon (CAST) system with a donor DNA comprising at least one engineered transposon end sequence and / or at least one integration co-factor protein. More particularly, the present disclosure provides systems comprising: an engineered CAST system or one or more nucleic acids encoding the engineered CAST system, wherein the CAST system comprises at least one or all of: i) at least one Cas protein, ii) one or more transposon-associated proteins, iii) at least one guide RNA (gRNA) complementary to at least a portion of a target nucleic acid sequence; and a donor nucleic acid comprising a cargo nucleic acid sequence flanked by at least one engineered transposon end sequence and / or at least one integration co-factor protein, or a nucleic acid encoding thereof.

Provided is a simple and efficient method of plant genome editing that does not involve the incorporation of a gene cassette for the expression system of a site-specific DNA modification protein. The method of the present invention comprises: (i) a step for mixing plant cells or a tissue containing plant cells together with a site-specific DNA modification protein and a needle-shaped inorganic compound in a liquid medium and making a disturbance to perforate the cells with the needle-shaped inorganic compound, thereby introducing the site-specific DNA modification protein into the cells; and (ii) a step for culturing the plant cells or the tissue containing the plant cells, into which the site-specific DNA modification protein has been introduced in step (i), thereby causing a DNA mutation specific to a target site in the genome of the plant cells.

The invention relates to a method for detecting DNA modification, in particular to a construction method of a DNA library with modified nucleotide, a detection method, a kit set and application. According to the scheme provided by the invention, the necessity of designing degenerate primers is avoided, the design difficulty of multiple primers is greatly reduced, the specificity of multiple PCR amplification is improved, and the formation of primer dimers is reduced.

To provide methods of profiling gene and protein expression of a plurality of nuclei from a single cell type.SOLUTION: Provided is a method of profiling nuclear epigenetic expression and / or nuclear structure, the method comprising: (a) processing a tissue sample containing a plurality of cell types to expose nuclei, the tissue sample comprising brain tissue, brain stem tissue, and / or spinal cord tissue; (b) contacting the nuclei with two or more affinity labels that bind to different nucleic acid transcripts or proteins that are specific to the nuclei of a cell type in the tissue sample to label the nuclei, the nucleic acid transcripts or proteins being encoded by genes selected from a specific group; (c) purifying the nuclei labeled with two or more affinity labels; and (d) (i) collecting information regarding histone modifications, binding of transcription factors, and DNA modifications for the labeled nuclei to profile the epigenetic expression of the nuclei, and / or (ii) collecting information on the chromosomal structure of the labeled nuclei to profile the nuclear structure.SELECTED DRAWING: Figure 1

The invention discloses a DNA modification and positioning method based on quantum dots, and relates to the technical field of DNA sequencing, and the method comprises the following steps: S1, combining CdSe quantum dots at the tail end of DNA to form a DNA and quantum dot compound; s2, enabling the DNA and quantum dot compound to pass through a nanopore of a nanopore detection system under the driving of an electric field, and collecting a tunneling current signal in real time by utilizing a current amplifier of the nanopore detection system; s3, performing filtering and noise reduction processing on the acquired tunneling current signal to obtain a processed current signal; s4, the processed current signals are subjected to feature recognition, the current base type and a recognition result are obtained, and the recognition result is recognition accuracy or recognition inaccuracy. According to the invention, the signal-to-noise ratio of single-base identification is improved, and the accuracy and stability of base identification are improved.

The present invention relates to a method of profiling covalent DNA modification(s) at a cellular or nuclei level comprising the steps of: providing a sample solution, comprising cell(s) comprising a nucleus comprising genomic DNA; adding a buffer comprising a nucleosome depletion agent to expose the genomic DNA; adding a fusion protein comprising a DNA modification binding domain and a nuclease part; incubating the fusion protein together with the exposed genomic DNA under conditions allowing the fusion protein to bind to the genomic DNA; washing the solution of step (d) with buffer to remove any excess fusion protein; incubating the solution of step (e) under conditions allowing the fusion protein to cut the genomic DNA in proximity of the DNA modification(s); and determining the sequence of the fusion protein incubated DNA of step (e) by high-throughput sequencing, thereby identifying the position(s) of the DNA modification(s) of the genomic DNA that have been sequenced. In other aspects, the invention also relates to a kit for use in performing the method.

Provided herein are compositions and methods for the multiplexed profiling of RNA and DNA modifications across transcriptomes and genomes, respectively. The methods combine molecular recognition of non-canonical features (e.g., base modifications, backbone modifications, lesions, and / or structural elements) of a target nucleic acid with a step of writing the information from this recognition event into the neighboring genetic sequence of the target nucleic acid using a barcode. The resultant barcoded nucleic acids are then converted into sequencing libraries and read by DNA / RNA sequencing methods. This step reveals the sequence of the barcode, which is correlated with the non-canonical feature in the target nucleic acid(s). The high throughput profiling methods described herein allow for identification and / or localization of one or more modifications in a target nucleic acid. The methods also allow for identification of the nature and location of several or all DNA / RNA modifications in parallel.

Provided herein are methods and compositions for use in detecting on-target and / or off-target cleavage of genomic DNA in a cell by a nuclease. The on-target and off-target cleavage is detected by using oligonucleotides that bind to the single-stranded 3′-overhang created at the cleavage site. The nuclease may be an engineered nuclease comprising a nucleic acid binding domain that binds to a target nucleic acid of interest.

The present invention relates to a composition comprising a modified DNA identifier sequence (MoDIS) comprising (i) a verification code nucleotide sequence (VC), (ii) a random index code nucleotide sequence (RIC) and (iii) an annealing site nucleotide sequence (AS) for specific binding to a primer for PCR amplification, wherein (a) AS is linked to an affinity tag or label and / or affinity tag compound for identifying, enriching and / or purifying MoDIS-bound nucleotide sequences; and (b) the nucleotide at the 5 '-terminus of the MoDIS comprises a 5'-azido-modified moiety or a 5 '-alkynyl-modified moiety.

The invention belongs to the technical field of DNA modification site detection, and particularly discloses a sequencing method for identifying a zooxanthellae genome 5-hydroxymethyl uracil based on double-enzyme cascade. The method comprises the following steps: specifically recognizing and cutting 5-hydroxymethyl uracil through human single-chain selective single-function uracil glycosylase to generate a base removal site, efficiently cutting an AP site by combining with cutting mismatched nuclease optimized by a catalytic structural domain, and directionally generating a 200-300bp DNA fragment containing an adhesive tail end; and analyzing the cleavage site by using next-generation sequencing to realize single-base resolution positioning. According to the method, cross interference and DNA damage of a traditional chemical oxidation method to 5hmC / 5fC are avoided through a double-enzyme cascade reaction, and single-base resolution positioning can be achieved; meanwhile, according to the method, the experimental process is simplified, 5hmU recognition, AP site generation and cutting are integrated into one-step reaction, and the library building period is shortened. Accurate positioning and quantitative analysis of the single base level of 5hmU are realized in a zooxanthellae genome, and epigenetic regulation and environmental stress response of the zooxanthellae are disclosed.

The present disclosure provides a DNA-targeting RNA that comprises a targeting sequence and, together with a modifying polypeptide, provides for site-specific modification of a target DNA and / or a polypeptide associated with the target DNA. The present disclosure further provides site-specific modifying polypeptides. The present disclosure further provides methods of site-specific modification of a target DNA and / or a polypeptide associated with the target DNA The present disclosure provides methods of modulating transcription of a target nucleic acid in a target cell, generally involving contacting the target nucleic acid with an enzymatically inactive Cas9 polypeptide and a DNA-targeting RNA. Kits and compositions for carrying out the methods are also provided. The present disclosure provides genetically modified cells that produce Cas9; and Cas9 transgenic non-human multicellular organisms.

The DNA modification kit comprises a kit body, one side of the inner wall of the kit body is fixedly connected with a fixing plate, circular grooves are formed in the top surface of the fixing plate at equal intervals, test tubes are inserted into the circular grooves, a sliding rod is fixedly installed in the middle of the inner bottom wall of each circular groove, and one side of the surface of each sliding rod is fixedly connected with a circular piece. A spring is fixedly connected to the top of the circular piece, a sliding block is slidably connected to the surface of the sliding rod, a special-shaped sliding groove is formed in the front face of the sliding block, and a connecting block is fixedly connected to one side of the sliding rod. The fixing plate, the test tubes, the springs, the driving assemblies and the special-shaped sliding grooves are used in cooperation, when medical staff need to fix the test tubes, the staff can place the specified test tubes in the circular grooves, the bottoms of the test tubes abut against the top of a circular disc, sliding blocks slide downwards along sliding rods, and the test tubes are fixed through the sliding rods. And then when the spring contracts, the driving rod can be matched with the sliding block to slide from the bottommost part of the special-shaped sliding groove to the topmost part for clamping.

A method of detecting an epigenetic DNA modification is described herein, the method comprising: attaching to DNA a labeling agent selective for the DNA modification; contacting an aqueous solution comprising the DNA with at least one addressable region on a surface of a substrate, wherein the addressable region comprises a positively charged substance capable of attaching the DNA to the surface, and the addressable region is surrounded by a hydrophobic region; and determining an amount of labeling agent in the addressable region. Further described herein is a kit comprising a substrate comprising discrete hydrophilic regions separated by a hydrophobic region, and a labeling agent selective for an epigenetic DNA modification; as well as an article of manufacture comprising a substrate comprising a discrete regions separated by a hydrophobic region, a positively charged substance and DNA attached thereto, the DNA comprising such a labeling agent.

This invention provides a DNA modification detection method using gel electrophoresis coupled with liquid chromatography-mass spectrometry (LC-MS / MS), comprising the following steps: separating a DNA sample to be tested by gel electrophoresis to obtain a gel strip containing the target DNA, wherein the electrophoresis buffer used in the gel electrophoresis method does not contain EDTA; enzymatically digesting the target DNA in the gel strip into single deoxynucleosides / deoxynucleotides by in-gel DNA digestion to obtain enzymatic digestion products; and detecting the enzymatic digestion products by liquid chromatography-mass spectrometry to obtain the results of DNA chemical modifications (including epigenetic modifications and DNA damage adducts). The detection method of this invention can accurately quantify the chemical modification information in different DNA components separated by gel electrophoresis.

The invention relates to a method for preparing a metasurface structure based on silicon dioxide microspheres and a DNA origami technology, which comprises the following steps: carrying out DNA modification on a gold film used in an experiment, and depositing a layer of ssDNA on the surface of the gold film; the preparation method comprises the following steps: forming a single-layer SiO2 microsphere structure on a gold film substrate modified with ssDNA through a Czochralski method, performing plasma treatment after the substrate is completely dried, putting the substrate into a constant-temperature and constant-humidity chamber for incubation, performing ultrasonic stripping on SiO2 microspheres in ultrapure water to form regular ssDNA array sites, depositing a pre-formed DNA origami structure or metal nanoparticles on the surface of a gold film, and drying the gold film to obtain the single-layer single-layer SiO2 microsphere structure. Under the action of complementary base pairing, the origami structure or the metal nanoparticles are accurately adsorbed on the array sites, and the surface of the gold film is cleaned to remove the non-specific binding origami structure or the metal nanoparticles, so that the metasurface structure is formed. The method is simple in process, low in equipment and environment requirements and low in preparation cost, and a paper folding structure can form various nano structures or various metal particles on the surface to form various complex metasurface structures such as a plasma structure and a chiral structure.

Provided herein are compositions and methods for the multiplexed profiling of RNA and DNA modifications across transcriptomes and genomes, respectively. The methods combine molecular recognition of non-canonical features (e.g., base modifications, backbone modifications, lesions, and / or structural elements) of a target nucleic acid with a step of writing the information from this recognition event into the neighboring genetic sequence of the target nucleic acid using a barcode. The resultant barcoded nucleic acids are then converted into sequencing libraries and read by DNA / RNA sequencing methods. This step reveals the sequence of the barcode, which is correlated with the non-canonical feature in the target nucleic acid(s). The high throughput profiling methods described herein allow for identification and / or localization of one or more modifications in a target nucleic acid. The methods also allow for identification of the nature and location of several or all DNA / RNA modifications in parallel.

To provide a DNA-targeting RNA that comprises a targeting sequence and provides, together with a modifying polypeptide, site-specific modification of a target DNA and / or a polypeptide associated with the target DNA.SOLUTION: Provided is a DNA-targeting RNA comprising: (i) a first segment comprising a nucleotide sequence complementary to a sequence in the target DNA; and (ii) a second segment that interacts with a site-directed modifying polypeptide, where, as an aspect, the first segment comprises 8 nucleotides having 100% complementarity to a sequence in the target DNA.SELECTED DRAWING: None

Aspects of the application relate to template nucleic acids capable of introducing edits into DNA targets. Templates of the application comprise two homology arms flanking a heterologous nucleic acid comprising nucleotides that do not base pair with a sequence in a DNA target. The nucleotides that do not base pair with the DNA target can be utilized to, for example, reduce recombination between the heterologous nucleic acid and the DNA target relative to recombination between the homology arms and the DNA target, which allows for site-specific insertion of one or more genetic edits. In some aspects, templates are described that can be used to introduce edits at a site that is separated from a DNA break by tens, hundreds, thousands, or more than thousands of nucleotides. Further aspects of the application relate to recombinant adeno-associated virus particles and methods related to template nucleic acids described herein.