386 results about "Cytosine" patented technology

The invention provides structure-oriented excavated cytosine deaminase with high editing activity and no sequence preference and application of the structure-oriented excavated cytosine deaminase. The invention discloses a series of novel cytosine deaminase. The cytosine deaminase disclosed by the invention has enhanced targeted editing performance; the enhanced targeted editing performance includes improved sequence-free preference, improved editing efficiency, reduced off-target effect, and improved targeted specificity.

The instant specification provides for evolved base editors which overcome deficiencies of those in art (including increased efficiency and / or decreased requirement for specific sequence-context at an editing site) and which are obtained a result of a phage-assisted continuous evolution (PACE) system. In particular, the instant specification provides for evolved cytidine base editors (e.g., based on APOBEC1, CDA, or AID cytidine deaminase domains) which overcome deficiencies of those in art (including increased efficiency and / or decreased requirement for specific sequence-context at an editing site) and which are obtained a result of a phage-assisted continuous evolution (PACE) system.

The invention relates to a metabolic marker for diagnosing diabetic secondary osteoporosis and application of the metabolic marker, and belongs to the technical field of biological medicine. The method comprises the following steps: firstly, screening out obviously different metabolites between osteoporosis (DOP) and osteoporosis-free groups (DM) in diabetic patients; and secondly, screening out significant difference metabolites between the primary osteoporosis patient and the healthy control group, and excluding the intersection of the two groups of difference metabolites to obtain metabolites which exclude the influence of the primary osteoporosis. Metabolic markers, namely cytosine nucleotide, ketoglutaric acid and triethylamine, are obtained through further screening, and when the three metabolic markers are independently used, AUCgt; the AUC during combined use is 0.99, so that the limitation that the sensitivity of the traditional bone mineral density detection on the osteoporosis recognition in the diabetic population is insufficient is effectively made up.

The invention belongs to the field of gene engineering, and relates to a DNA binding protein based on TraC effect protein, a fusion protein with a cytosine / adenine base editing function, an editing system and nucleic acid molecules or constructs for encoding the DNA binding protein and the fusion protein. The invention also relates to complexes and kits for gene editing comprising the fusion proteins of the invention or the nucleic acid molecules encoding them. The base editing tool disclosed by the invention can realize efficient, accurate and safe base editing in cells of various eukaryotes such as animals and plants. The unique characteristics of the base editing tool enrich the types of existing base editing tools, and the base editing tool has wide application prospects.

Provided herein are systems, compositions, and methods for generating immunogenic peptides or epitopes from tumor associated antigens (e.g., in vivo or ex vivo). Polynucleotides (e.g., genes) encoding the tumor associated antigens may be edited at selected target sites by nucleobase editors comprising a catalytically-inactive Cas9 and a cytosine deaminase, leading to the expression of heteroclitic or cryptic peptides that are more immunogenic than the native peptide derived from the tumor associated antigens. The heteroclitic or cryptic peptide elicit strong tumor-specific immune response (e.g., T-cell response or B-cell response), which inhibits tumor growth and metastasis.

The invention discloses cytosine deaminase as well as a related biological material and application thereof, and belongs to the technical field of protein. The technical problem to be solved by the invention is to excavate natural cytidine deaminase without sequence preference and improve the base editing efficiency and range. The invention provides a 15th-143rd site protein of which the amino acid sequence is SEQ ID NO.2. The invention also provides a preparation method of the protein. The invention also provides application of the protein and related biological materials thereof in single base editing. According to the application, metagenome big data is utilized, comprehensive and systematic information mining analysis is carried out on novel cytidine deaminase, and a protein L85 which is highly similar to a known DddA protein structure but low in sequence similarity is selected from the novel cytidine deaminase for functional verification. The protein L85 has high catalytic activity as cytidine deaminase, is not limited by target sequence preference, can be widely applied to gene editing in vitro and in cells / tissues, and has good application potential and development value.

The present invention provides for novel methods for regulating and detecting the cytosine methylation status of DNA. The invention is based upon identification of a novel and surprising catalytic activity for the family of TET proteins, namely TET1, TET2, TEM3, and CXXC4. The novel activity is related to the enzymes being capable of converting the cytosine nucleotide 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation.

The present invention relates to the field of genetic engineering. Specifically, the present invention relates to cytosine deaminase and use thereof in base editing. More specifically, the present invention relates to a base editing system based on a newly identified cytosine deaminase, a method for base editing a target sequence in the genome of an organism (e.g., a plant) using the base editing system, and a genetically modified organism (e.g., a plant) produced by the method and progenies thereof.

The present invention relates to a fusion protein that produces point mutations in cells, its preparation and use. Specifically, the fusion protein provided by the present invention contains a Cas enzyme that lacks cytosine deaminase and nuclease activity and retains helicase activity, or is formed by a Cas enzyme that lacks cytosine deaminase and nuclease activity and retains helicase activity. The present invention also relates to the coding sequence of the fusion protein, a polynucleotide sequence containing the coding sequence, a nucleic acid construct containing the polynucleotide sequence, a corresponding host cell, a method for producing point mutations in a cell, and a kit, etc. By using the present invention, it is possible to achieve site-directed mutagenesis while obtaining high mutation efficiency and a variety of mutation combinations in a specific gene region.

The invention discloses an information storage method based on DNA (deoxyribonucleic acid) coding, which comprises the following steps: converting digital information to be stored into a quaternary code, converting the quaternary code into a DNA sequence consisting of adenine A, cytosine C, guanine G and thymine T according to a preset mapping rule, and expressing an additional information dimension by using a chemical modification state of a nucleotide pair; inserting error detection and correction codes in the DNA sequence every a predetermined number of basic group positions, segmenting the encoded DNA sequence into a plurality of fragments with the length of 100-150bp, and adding a specific recognition sequence and an index marker at two ends of each fragment; a reversible thermosensitive response DNA nanostructure is used as a carrier, DNA fragments are selectively combined to the carrier, and hierarchical storage and rapid retrieval of information are realized through a temperature gradient control system; the DNA storage information density can be remarkably improved, the service life of DNA storage can be remarkably prolonged, and a novel technical path is provided for large-scale, long-term and safe molecular information storage.

The invention provides a non-disease diagnosis purpose 5-hydroxymethylcytosine 5hmC detection method and a non-disease diagnosis purpose 5-hydroxymethylcytosine 5hmC detection kit. The detection method comprises the following steps: S1, glycosylation treatment; s2, carrying out enzyme digestion treatment on the GSREs; s3, carrying out APOBEC deamination treatment; s4, carrying out hydroxymethylation detection; the kit comprises a glycosylation reaction kit, a GSREs enzyme digestion reaction kit, an APOBEC deamination reaction kit and a PCR (Polymerase Chain Reaction) kit. The glycosylation reaction kit comprises a CutSmart buffer solution, UDP-Glucose, a T4-BGT enzyme and nuclease-free water, wherein the CutSmart buffer solution, the UDP-Glucose and the T4-BGT enzyme are used as raw materials; the GSREs enzyme digestion reaction kit comprises a GSREs enzyme, an enzyme digestion reaction buffer solution and nuclease-free water, the APOBEC deamination reaction kit comprises APOBEC, BSA (Bovine Serum Albumin), an APOBEC Reactive buffer solution and nuclease-free water; the PCR kit comprises a PCR reaction solution, a primer and a probe. According to the 5-hydroxymethylcytosine 5hmC detection method and kit for non-disease diagnosis purpose provided by the invention, precise quantification of extremely high specificity of 5hmC is realized, and the detection sensitivity is also improved while the specificity of a detection signal is improved.

The invention provides a perovskite solar cell, a preparation method thereof and a photovoltaic module. The perovskite solar cell comprises a first electrode, a hole transport layer, a perovskite layer, an electron transport layer and a second electrode which are stacked in sequence. The material of the hole transport layer comprises a biomolecular material which is fixed on a self-assembly monomolecular layer material through an intermolecular force, and the biomolecular material comprises one or more of guanine, adenine, thymine, uracil, cytosine, hydroxymethylcytosine, xanthine, hypoxanthine, 6-mercaptopurine and corrin. According to the perovskite cell provided by the invention, the biomolecular material with a plurality of nitrogen heterocyclic structures and amino groups is added to generate multipoint hydrogen-bond interaction and pi-pi accumulation with the tail end of the SAM, so that the orderliness of molecular arrangement is improved, and the dipole direction and strength of the whole interface are regulated and controlled; and a coordination bond or hydrogen bond network is formed by the coordination compound and uncoordinated cations or anions in perovskite, so that interface defects are effectively passivated, and the thermal stability and the humid and hot life of a device are enhanced.

The present invention discloses a recombinant glycosylase base editing system and its application. The recombinant glycosylase base editing system of the present invention is an APOBEC-nCas9-Ung1 or APOBEC(R33A)-nCas9-Rad51-Ung1 base editing system; the base editing system APOBEC-nCas9-Ung1 includes cytosine deaminase APOBEC, nuclease nCas9 and uracil DNA glycosylase Ung1; the base editing system APOBEC(R33A)-nCas9-Rad51-Ung1 includes cytosine deaminase APOBEC(R33A), nuclease nCas9, single-stranded DNA binding protein Rad51 and uracil DNA glycosylase Ung1. Experiments have shown that the recombinant glycosylase base editing system of the present invention can improve the editing efficiency of base C to base G in the genome sequence of an organism or a biological cell and the purity of the editing product.

The invention relates to the field of gene engineering. In particular, the present invention relates to cytosine deaminases and their use in base editing. More specifically, the invention relates to a method for screening and identifying a deaminase, a base editing system based on a newly identified cytosine deaminase, a method for editing a target sequence in a genome of an organism (such as a plant) by using the base editing system, and a method for screening and identifying the target sequence. As well as genetically modified organisms (e.g., plants) and progeny thereof produced by the method.

The invention comprises a process for the production of a linear P-linked oligonucleotide bearing an alkyl phosphonate group at 5′-terminus of the formula Iwherein R1 is a C1-4-alkyl group, R2 is fluoro, hydroxyl or C1-4-alkoxy, X is sulfur or oxygen, the term Nucleobase stands for an optionally modified adenine, cytosine, guanine or uracil and the term oligo strand stands for the remaining P-linked oligo nucleotide strand, and wherein the oligo strand comprises at least one uracil nucleobase.The process is performed under conditions that the level of alkyl transfer impurities and the level of cyanoethyl (CNET) impurities in the linear P-linked oligonucleotide of formula I, is substantially reduced.

Described herein are nucleic acid molecules, compositions, recombinant AAV (rAAV) particles, kits, and methods for delivering a base editor (or a "nucleobase editor") to a cell, for example via an AAV vector. In particular, the present disclosure provides compositions, methods and uses for delivery of adenine base editors and cytosine base editors in a single AAV vector (or genome). Further described herein are improved AAV vectors comprising a minimised size regulatory component, for example, a regulatory component that can enable packaging of a base editor. Provided herein are methods and compositions for delivering a base editor protein to a cell or tissue in a single recombinant AAV (rAAV) vector. Improved methods and compositions for in vivo delivery of these base editors in a single rAAV particle are contemplated herein. Further provided herein are base editors, and compositions and cells comprising these base editors.

The disclosure provides methods for processing nucleic acid populations containing different forms (e.g., RNA and DNA, single-stranded or double-stranded) and / or extents of modification (e.g., cytosine methylation, association with proteins). These methods accommodate multiple forms and / or modifications of nucleic acid in a sample, such that sequence information can be obtained for multiple forms. The methods also preserve the identity of multiple forms or modified states through processing and analysis, such that analysis of sequence can be combined with epigenetic analysis.

The disclosure provides methods for processing nucleic acid populations containing different forms (e.g., RNA and DNA, single-stranded or double-stranded) and / or extents of modification (e.g., cytosine methylation, association with proteins). These methods accommodate multiple forms and / or modifications of nucleic acid in a sample, such that sequence information can be obtained for multiple forms. The methods also preserve the identity of multiple forms or modified states through processing and analysis, such that analysis of sequence can be combined with epigenetic analysis.

The disclosure provides methods for processing nucleic acid populations containing different forms (e.g., RNA and DNA, single-stranded or double-stranded) and / or extents of modification (e.g., cytosine methylation, association with proteins). These methods accommodate multiple forms and / or modifications of nucleic acid in a sample, such that sequence information can be obtained for multiple forms. The methods also preserve the identity of multiple forms or modified states through processing and analysis, such that analysis of sequence can be combined with epigenetic analysis.

The invention discloses a Cg12n-v4.6 nuclease. The amino acid sequence of the nuclease is as shown in SEQ ID No. 12. The invention also discloses a preparation method of the nuclease. The Cg12n-v4.6 nuclease mutant with increased activity and improved editing efficiency is developed, and the editing efficiency of the Cg12n-v4.6 nuclease mutant is up to 70% or above. Meanwhile, through engineering modification of corresponding sgRNA of Cas12n, the size of the sgRNA skeleton is reduced. An enhanced Cas12n gene editing tool, namely an eCg12n gene editing system, is developed by combining an engineered Cg12n-v4.6 nuclease mutant and a simplified sgRNA skeleton. Meanwhile, by fusing deaminase, a compact cytosine base editor and a compact adenine base editor are constructed, and the cytosine base editor is applied to edit and induce disease genes to generate early termination codons, so that safe knockout of the genes is realized. Compared with an original wild type editing system, the Cg12n-v4.6 nuclease mutant and a compact eCg12n gene editing system derived from the Cg12n-v4.6 nuclease mutant have the advantages that the editing efficiency is improved, the system size is further simplified, and the Cg12n-v4.6 nuclease mutant and the compact eCg12n gene editing system derived from the Cg12n-v4.6 nuclease mutant are more suitable for gene editing application.

The invention provides an efficient double-base editing system without PAM (Pulse Amplitude Modulation) limitation. According to the technology, different point mutations are introduced into SpRY, the SpRY is fused with efficient adenine deaminase and cytosine deaminase to construct a double-base editor, and efficient PAM-limitation-free double-base editing is achieved in the rice protoplast. According to the plant basic group editing method, efficient and PAM-less double basic group editing can be achieved, and the plant basic group editing technology is wider in target range, higher in efficiency and higher in double basic group editing product proportion.

The present disclosure generally relates to an evolved cytidine deaminase derived from a cytidine deaminase, and a method of editing DNA using the same. In some aspects, the present disclosure describes directed evolution of a TadA-derived adenosine deaminase (TadA-CD) for cytidine deamination. In some embodiments, the TadA-CD comprises a plurality of mutations as compared to the parent TadA variant. In some embodiments, the TadA-CD is fused to a programmable DNA binding protein. Other aspects of the present disclosure generally relate to a cytosine base editor (CBE) comprising a programmable DNA binding protein and a TadA-CD. In some embodiments, the disclosed cytosine base editor has an improved conversion efficiency and a reduced off-target editing frequency compared to a naturally occurring CBE. Polynucleotides, vectors, and kits for generating and delivering CBE are also provided. Also provided are cells containing such vectors and CBE. In addition, methods of treatment comprising administration of CBE are also provided.

The invention provides a plant cytosine single-base editor as well as a construction method and application thereof, and relates to the technical field of plant genetic engineering. The single-base editor provided by the invention is named as hyPopCBE, and has a structural formula shown in the description, the editor comprises hA3A / Y130F cytosine deaminase, an optimized human-derived Rab51 single-stranded DNA binding protein structural domain, a fusion protein of nCas9 nuclease and a uracil glycosylase inhibitor, a fusion protein expression unit of phage MS2 coat protein and the uracil glycosylase inhibitor, and a guide RNA transcription expression unit integrated with an MS2 binding sequence. Compared with an existing plant cytosine single-base editor, the single-base editor constructed by the method can remarkably improve the editing efficiency of plant alleles, and the stability is higher; when the herbicide-resistant poplar gene is applied to poplar, a new herbicide-resistant poplar germplasm can be successfully created, and a technical support is provided for research and molecular breeding of a poplar functional genome.

Disclosed herein are methods, compositions, and devices for use in early detection of cancer. The claimed methods include the detection of minimal residual disease (MRD) by analysis of urine samples. The method may involve the use of methyl binding domain proteins or by direct sequencing and may involve detection of somatic variants or detection of methylated cytosines. Also claimed is a method which determines a cancer status by generating an input vector based on quantitative measures created from urine sequence reads and analysing the input vector with a machine learning based model.

A method of detecting the level of 5-hydroxymethylcytosines (5-hmc) in a DNA molecule of a cell having a 5-hmc prevalence lower than 0.002% of total DNA bases is provided. The method comprising:(a) attaching a 5-hmc labeling agent to the DNA molecule; and(b) subjecting the DNA molecule to an imaging method suitable for detecting the labeling agent, thereby detecting the level of 5-hmc in the DNA molecule. Also provided is a method of diagnosing cancer in a subject in need thereof, the method comprising:(a) providing a DNA sample of a cell of the subject;(b) detecting the level of 5-hmc in the DNA sample as described herein; wherein a significant decrease in the level of 5-hmc in the DNA sample, as compared to a control DNA sample from a healthy subject is indicative that the subject has cancer.

The present invention belongs to the technical field of genetic engineering. Provided are a single-base editor, a deaminase used therein, and the use thereof. The technical problems to be solved are to identify a naturally occurring cytosine deaminase without sequence preference, construct a base editor, and improve the efficiency and scope of base editing. In order to solve the technical problems above, a cytosine base editor is provided. The cytosine base editor is a fusion protein, wherein the fusion protein is a protein containing a cytidine deaminase, a Cas protein and a uracil-DNA glycosylase inhibitor, and the cytidine deaminase is a protein having an amino acid sequence of positions 28-164 of SEQ ID NO. 2. Further provided is the use of the fusion protein above and a biomaterial related thereto in plant single-base editing. The single-base editor can improve the efficiency of cytosine base editing and accurately mediate the base mutation of a target, and is widely applicable in the cells of maize and even other plants.

The invention discloses a single-base editor, deaminase used by the single-base editor and application of the single-base editor, and belongs to the technical field of genetic engineering. The technical problems to be solved in the invention are as follows: natural cytosine deaminase without sequence preference is mined, a base editor is constructed, and the base editing efficiency and range are improved. In order to solve the technical problem, the application provides a cytosine base editor, the cytosine base editor is a fusion protein, the fusion protein is a protein containing cytidine deaminase, a Cas protein and a uracil glycosylase inhibitor, and the cytidine deaminase is a protein at the 28th-164th site of an amino acid sequence SEQ ID NO.2. The invention also provides application of the fusion protein and related biological materials thereof in plant single base editing. The single-base editor provided by the invention can improve the efficiency of cytosine base editing, can accurately mediate the base mutation of a target site, and can be widely applied to corn and even other plant cells.

Methods of predicting a chromatin state metric associated with a genomic region in a sample are provided. The methods comprise: receiving sequence data comprising genetic data and epigenetic data indicative of the presence of one or more epigenetic bases including methylated cytosine and hydroxymethylated cytosine at one or more genomic positions, the one or more epigenetic bases, the genetic data and epigenetic data obtained from a single assay; and predicting for each base or set of bases of the genomic region and using the sequence data associated with the genomic region, a value of the chromatin state metric, wherein the predicting is performed using a machine learning model.

The invention relates to a T7RNA polymerase-guided bacillus subtilis cytosine base editor and application thereof, and belongs to the technical field of gene editing. A T7RNAP guided cytosine base editor is developed in bacillus subtilis, four kinds of cytidine deaminase from different sources are selected to be fused with T7RNAP, the cytidine deaminase is fused at the N end of the T7RNAP, and a fusion body with the highest mutation activity is obtained through screening; the cytosine base editor is used for evolving a global transcriptional regulatory factor CodY, in-situ evolution of genome protein is achieved, and the expression level of bovine-derived beta-lactoglobulin (beta-Lg) is improved. According to the cytosine base editor, the operable range of the cytosine base editor on a genome is widened, multi-site base (C-T) editing can be realized, and an editing window is large.

The disclosure provides methods for processing nucleic acid populations containing different forms (e.g., RNA and DNA, single-stranded or double-stranded) and / or extents of modification (e.g., cytosine methylation, association with proteins). These methods accommodate multiple forms and / or modifications of nucleic acid in a sample, such that sequence information can be obtained for multiple forms. The methods also preserve the identity of multiple forms or modified states through processing and analysis, such that analysis of sequence can be combined with epigenetic analysis.