34 results about "Splice Site SNP" patented technology

The invention belongs to the field of biological medicine, and relates to a guide agRNA for regulating RNA splicing. The invention provides an application of a guide agRNA (Ribonucleic Acid) in preparation of a medicine for changing expression of a target protein by cells of a subject. The guide agRNA recruits ADAR protein to the 3 'splice site or pseudo 3' splice site to edit the A base of the 3 'splice site or pseudo 3' splice site, thereby splicing the whole exon located at the 3 'splice site or flanking the pseudo 3' splice site of the intron from the precursor mRNA, thereby changing the level of mRNA encoding the target protein, and altering the expression of the target protein in the cell. The target gene pre-mRNA selective splicing is regulated and controlled to influence the function or expression of the functional RNA and the target protein of the target gene, so that the purpose of treating diseases is achieved.

Disclosed herein is a promoter sequence comprising at least a portion of a ABCB4 promoter sequence or reverse complement thereof, wherein the splice sites of the ABCB4 promoter are mutated, and lentiviral vector sequences comprising said promoter sequences operably linked to a transgene. Preferably, the promoter sequence and transgene are present in a reverse expression cassette in reverse orientation to the direction to the lentiviral vector sequence. When a transgene for ABCB4 is used, the lentiviral vector can be used in the treatment of PFIC3.

The present invention discloses a circular gRNA and its related biomaterials and applications. The coding gene of the circular gRNA includes a 3' intron, a 3' splice site, a gRNA coding gene, a 5' splice site and a 5' intron in sequence, wherein the 3' splice site sequence is as shown in positions 2810-2814 of SEQ ID No.1, and the 5' splice site sequence is as shown in positions 2917-2930 of SEQ ID No.1. The circular gRNA is not attacked by nucleases, thereby improving the stability of the CRISPR / cas9 system.

A method and compound for regulating SCN1A gene mRNA and protein expression. Provided is a use of a guide agRNA in preparation of a drug for altering expression of a target protein in a cell of a subject. The cell has a precursor mRNA encoding the target protein, wherein the precursor mRNA comprises an intron, an exon flanking a 5' splice site of the intron, and an exon flanking a 3' splice site or a pseudo 3' splice site of the intron; the guide agRNA is capable of binding to the precursor mRNA to form a structure capable of recruiting ADAR in the cell, thereby forming a complex containing an ADAR protein; and the target protein is a Nav1.1 protein. By regulating alternative splicing of a target gene pre-mRNA, the functional RNA of the target gene and the function or expression of the target protein are affected, thereby achieving the objective of disease treatment.

A method for regulating the mRNA and protein expression of an SCN1A gene, and a compound. Provided is a use of an antisense oligonucleotide guide RNA in the preparation of a drug for altering the expression of a target protein in a cell of a subject. The cell has a pre-mRNA encoding the target protein, wherein the pre-mRNA comprises an intron, an exon flanking a 5' splice site of the intron, and an exon flanking a 3' splice site or pseudo 3' splice site of the intron; the antisense oligonucleotide guide RNA can bind to the pre-mRNA to form a structure capable of recruiting ADAR in a cell, thereby forming a complex containing an ADAR protein; and the target protein is an Nav1.1 protein. That is, the function or expression of the functional RNA and target protein of a target gene is affected by regulating the alternative splicing of the pre-mRNA of the target gene, thereby achieving the objective of treating diseases.

The invention discloses a tumor tissue splicing recognition and neoantigen peptide screening method based on an ONT sequencing platform, and belongs to the technical field of tumor immunology. Comprising the following steps: extracting RNA from a tumor tissue, separating mRNA, constructing a sequencing library, and sequencing through an ONT sequencing platform to obtain original data; performing basic group identification on the original data to obtain fastq format data; comparing the data to a reference genome reconstruction transcript, identifying a variable splicing event based on a comparison result, and performing difference analysis; screening out variable splicing events with significant differences, and extracting novol transcript coding sequences related to the events; extracting a 9mer peptide fragment crossing a splicing site in a splicing boundary region of the transcript; performing MHC binding affinity scoring on the peptide fragments, and screening out candidate new antigen peptide fragments; and carrying out mass spectrum verification on the candidate new antigen peptide fragment, and screening the actually expressed peptide fragment. The method can be widely applied to tumor immunomarker screening, new antigen vaccine design and individualized treatment strategy development.

A targeted high-throughput sequencing method for detecting splice isoforms, including a method for establishing a sequencing library for high-throughput sequencing, comprising the following steps: 1) reverse transcription of sample RNA using a reverse transcription primer, adding common dNTPs and 3'-modified dNTPs to generate a first-strand cDNA; 2) ligating an oligonucleotide fragment with an alkyne modification at the 5' end to the cDNA fragment obtained in step 1) via a click chemistry reaction; 3) performing PCR amplification using the reaction product of step 2) as a template; and 4) obtaining a sequencing library. Targeted enrichment primers can be introduced during the reverse transcription or PCR amplification steps. The enrichment step reaction system contains multiple gene-specific primers, each designed based on the downstream exon segment of the alternative splicing event in the transcript, ensuring that the random-length fragments generated by reverse transcription cover the splice site.

A method of preparing a circular RNA includes transcribing a vector to form a precursor RNA, in which the vector includes the following elements operably connected to each other and arranged in the following sequence: a) a 5′ element, b) a 3′ Group I self-splicing intron fragment containing a 3′ splice site dinucleotide, c) none or an element containing an internal ribosome entry site (IRES) and a protein coding region or an element containing a noncoding region, d) a 5′ Group I self-splicing intron fragment containing a 5′ splice site dinucleotide, and e) a 3′ element, in which 5′ element and 3′ element form a stable structure with a Gibbs free energy (ΔG) from −190 kcal / mol to −9.0 kcal / mol, provided that the stable structure is not a duplex with at least 95% base pairing between 5′ element and 3′ element, in which 3′ Group I self-splicing intron fragment and 5′ Group I self-splicing intron fragment form a self-cleaving and self-ligating RNA molecule, thereby generating circular RNA.

The invention relates to the technical field of plant genetic engineering, in particular to a gene down-regulation method based on regulation of precursor mRNA (messenger Ribonucleic Acid) splicing efficiency, which comprises the following steps: firstly, identifying at least one intron 5'terminal splicing site containing a GT sequence in a target gene; aiming at the site, designing a guide RNA (Ribonucleic Acid) sequence capable of targeting the downstream fourth or fifth nucleotide, and verifying the cleavage activity of the guide RNA sequence; constructing a gene editing vector by using the guide RNA sequence which is verified to be effective and a Cas9 nuclease coding gene; introducing the vector into a plant cell, carrying out genetic transformation and regeneration to obtain a transformed plant, and screening out a mutant which is subjected to site-directed editing at a target site through DNA (Deoxyribose Nucleic Acid) sequencing; and finally, extracting RNA (Ribonucleic Acid) of the mutant, reversely transcribing into cDNA (Complementary Deoxyribonucleic Acid), and analyzing transcript change through PCR (Polymerase Chain Reaction) amplification and gel electrophoresis. According to the invention, stable, heritable and accurate knock-down of gene expression is realized, and a new tool is provided for essential gene function research and crop character improvement.

A member of the Solanum species, such as a tomato (Solanum lycopersicum) plant, is provided that exhibits a jointless phenotype, wherein the plant comprises a Jointless-2 (j2) allele that is defined by the removal or disruption of a splice site within the SlMBP21 (Solyc12g038510) gene The removal or disruption of a splice site results in the complete or partial loss of at least one exon in an mRNA transcribed from a SlMBP21 gene (Solyc12g038510). More particularly, the removal or disruption of a splice site results in the complete or partial loss of exon 5 in an mRNA transcribed from a SlMBP21 gene (Solyc12g038510). The removal or disruption of a splice site may be carried out using precision breeding techniques.

A method of preparing a circular RNA includes transcribing a vector to form a precursor RNA, in which the vector includes the following elements operably connected to each other and arranged in the following sequence: a) a 5′ element, b) a 3′ Group I self-splicing intron fragment containing a 3′ splice site dinucleotide, c) none or an element containing an internal ribosome entry site (IRES) and a protein coding region or an element containing a noncoding region, d) a 5′ Group I self-splicing intron fragment containing a 5′ splice site dinucleotide, and e) a 3′ element, in which 5′ element and 3′ element form a stable structure with a Gibbs free energy (ΔG) from −190 kcal / mol to −9.0 kcal / mol, provided that the stable structure is not a duplex with at least 95% base pairing between the 5′ element and 3′ element, in which 3′ Group I self-splicing intron fragment and 5′ Group I self-splicing intron fragment form a self-cleaving and self-ligating RNA molecule, thereby generating circular RNA.

The invention relates to the technical field of biology, in particular to a short-chain non-coding RNA molecular tool vector expression system and application thereof. The molecular tool vector expression system comprises an endothelial specific gene promoter sequence and a skeleton sequence for expressing short-chain non-coding RNA including snoRNA, the endothelial specific gene promoter sequence is derived from an ICAM2 gene, and the skeleton sequence is derived from a No.2 intron region of SNORD32A in an rpl13a gene; a traditional U6 or CMV promoter is replaced with an endothelial specific promoter derived from the ICAM2 gene, expression of short-chain non-coding RNA can be specifically started, a skeleton sequence derived from a second intron region in the rpl13a gene is designed, the skeleton sequence can be promoted to have a plurality of splicing sites, and the splicing sites can be used for promoting the expression of the short-chain non-coding RNA. Therefore, accurate, efficient and stable overexpression of the short-chain non-coding RNA including the snoRNA can be realized.

A modified U7 snRNA construct comprising at least one antisense sequence having between 16 to 30 nucleotides which are at least 90% complementary to a splicing element of a TDP-43 regulated cryptic exon sequence or flanking regions thereof and wherein the U7 snRNA construct is capable of modulating splicing of the TDP-43 regulated cryptic exon in a cell. The splicing element may be selected from a splice site, a TDP-43 binding region, or an exonic splice enhancer. Vectors and pharmaceutical compositions comprising the construct are also described, as well as constructs for use in the treatment of diseases associated with TDP-43 dysfunction. Example TDP-43 regulated cryptic exon sequences include TDP-43 regulated cryptic exons in UNC13A, STMN2 and INSR genes.

The present invention relates to a therapeutic oligonucleotide for use in treating a disease in a subject that is associated with or caused by a missense, nonsense or in-frame indel mutation in a gene of the genome of the subject, wherein the therapeutic oligonucleotide is capable of specifically hybridizing to the 5'-splice site, the 3'-splice site or an exonic splicing enhancer (ESE) of an out-of- frame exon in the gene with the missense, nonsense or in-frame indel mutation, thereby causing skipping of the out-of-frame exon.

A linear precursor RNA molecule is provided, having a 5' end and a 3' end, and comprising in the 5'-to-3' direction i) a first self-splicing sequence, ii) a sequence of interest, and iii) a second self-splicing sequence, wherein the linear precursor RNA molecule is capable of forming a circular RNA upon splicing of said first and second self-splicing sequences; wherein the first self-splicing sequence comprises a first homopolymeric moiety located between said 5' end and a splice site first self-splicing sequence, and / or wherein the second self-splicing sequence comprises a second homopolymeric moiety located between said 3' end and a splice site of the second self-splicing sequence. The invention may improve the production and / or purification of circular RNA.

The technology disclosed relates to splice site prediction and aberrant splicing detection. In particular, it relates to a splice site predictor that includes a convolutional neural network trained on training examples of donor splice sites, acceptor splice sites, and non-splicing sites. An input stage of the convolutional neural network feeds an input sequence of nucleotides for evaluation of target nucleotides in the input sequence. An output stage of the convolutional neural network translates analysis by the convolutional neural network into classification scores for likelihoods that each of the target nucleotides is a donor splice site, an acceptor splice site, and a non-splicing site.

Disclosed are methods and kits for eliminating cancer cells and treating cancers by targeting neo splice sites or cryptic exons of oncogenic gene fusions.

The embodiment of the invention discloses a gene splicing site recognition method and system based on small molecule regulation, and the method comprises the steps: obtaining the gene expression quantity of each candidate gene sequence, so as to select a plurality of reliable gene sequences; obtaining a candidate pseudo exon insertion event of each reliable gene sequence under the treatment of each small molecule compound, and selecting a reliable pseudo exon insertion event from a plurality of candidate pseudo exon insertion events; training a pre-constructed deep learning model according to the reliable gene sequence and the reliable pseudo exon insertion event to obtain a splicing site recognition model; obtaining a target gene sequence, and obtaining a base position feature sequence corresponding to the target gene sequence; and inputting the base position feature sequence into a splicing site identification model to predict the probability that each base is a splicing site through the splicing site identification model so as to obtain a splicing site coordinate corresponding to the target gene sequence according to the probability, thereby improving the splicing site identification accuracy.

Provided is a guide agRNA for regulating RNA splicing. Also provided is use of the guide agRNA in the preparation of a drug for changing the expression of a target protein by cells of a subject. The guide agRNA recruits the ADAR protein to the 3' splice site or pseudo-3' splice site to edit the base A of the 3' splice site or pseudo-3' splice site, so that an entire exon flanking the 3' splice site or pseudo-3' splice site of the intron is spliced from the precursor mRNA, thereby changing the level of the mRNA encoding the target protein and the expression of the target protein in the cells. That is, the selective splicing of the pre-mRNA of the target gene is regulated to affect the function or expression of the functional RNA and target protein of the target gene, achieving the purpose of treating diseases.

The present invention relates to an acceptor splice region, as well as uses and applications thereof.

Provided herein is a method for inhibiting abnormal splicing in a MicroTubular Assembly Protein-2 (STMN2) transcript, the method comprising: genetically editing an STMN2 gene in a cell to delete: (a) one or more nucleotides in a 3'splicing site of Intron 1, where the 3 'splicing site is adjacent to exon 2a; (b) one or more nucleotides in a region of intron 1 adjacent to the 3'splice site; or both (a) and (b), whereby the production of an STMN2 transcript comprising exon 2a is inhibited and the production of a functional STMN2 transcript is increased in the cell. The invention also provides a gene editing system for gene modification of the STMN2 gene.

This disclosure provides a DNA construct comprising: (a) a start exon encoding a start codon; (b) a first frameshift exon downstream of the start exon, the first frameshift exon including a first receptor splice site, wherein inclusion or exclusion of the first frameshift exon in alternative splicing determines the downstream reading frame of a mature mRNA transcript; and (c) a first specific exon downstream of the first frameshift exon, the first specific exon including a second receptor splice site, a first reporter gene, and a second reporter gene, wherein the first reporter gene is located upstream or downstream of the second reporter gene, and wherein the first reporter gene and the second reporter gene are in different reading frames, wherein exclusion of the first frameshift exon causes the first reporter gene to be in the same reading frame as the start codon, and wherein inclusion of the first frameshift exon causes the second reporter gene to be in the same reading frame as the start codon.

A peptide nucleic acid derivative of Formula I is provided to tightly bind to a splice site within a pre-mRNA in a sequence specific manner. Given with excellent cell membrane permeability and strong affinity for RNA, the peptide nucleic acid derivative induces exon skipping in cells treated with the peptide nucleic acid at sub-femtomolar concentration as “naked” oligonucleotide. The compound shows therapeutic activity in subjects upon systemic administration even at 1 μg / Kg or less, and therefore is useful to treat a disease or symptom at affordable treatment cost.

An HTT nucleic acid trans-splice molecule is described that includes a coding domain comprising one or more HTT exons, a splice site, and a binding domain that binds to a target intron of an HTT precursor mRNA. The HTT nucleic acid trans-splice molecules described herein may also be used in combination with, for example, an MSH3 binding domain aligned in series with an HTT binding domain, an MSH3 nucleic acid trans-splice molecule, an MSH3 splice modulator, an antisense oligonucleotide or antisense RNA for either of MSH3 or HTT, and an MSH3 or HTT microRNA (miRNA) and constructs encoding them. Also encompassed are compositions comprising the nucleic acid trans-splice molecules described herein, as well as compositions comprising a nucleic acid trans-splice molecule in combination with an additional therapeutic agent (e.g., an MSH3 nucleic acid trans-splice molecule, an MSH3 splice modulator, an antisense oligonucleotide or antisense RNA for any one of MSH3 or HTT). The nucleic acid trans-splicing molecules may be used alone or in combination with additional therapeutic agents in methods of treating Huntington's disease (HD). Also described herein are nucleic acid trans-splice molecules for use in the treatment of HD or in the preparation of medicaments for the treatment of HD, alone or in combination with additional therapeutic agents. Also encompassed herein are MSH3 nucleic acid trans-splice molecules, MSH3 splice modulators, and MSH3 miRNAs, as well as constructs encoding them, which can be used, alone or in combination and / or in combination with additional therapeutic agents, for the treatment of nucleotide repeat disorders (e.g., HD) or for the preparation of medicaments for the treatment of nucleotide repeat disorders (e.g., HD).

The invention provides a target gene expression quantity down-regulation method based on an intron cleavage site. The method comprises the following steps: acquiring large-scale transcriptome data, and analyzing the intron retention condition in a material based on the large-scale transcriptome data. And selecting a target intron, and carrying out micro mutation design on the target intron to obtain a mutant intron. And inserting the mutant intron and the target intron into a carrier containing GFP (Green Fluorescent Protein) to construct a splicing expression carrier. And transforming the splicing expression vector into the rice protoplast to obtain the protoplast containing the vector. According to the method, the intron 5 'splicing site (5' SS) intensity is finely adjusted, and intron retention is induced, so that stable down-regulation of target gene expression is realized, the problems of complete inactivation, lethal or phenotype abnormality caused by a traditional CRISPR / Cas9 knockout method are avoided, and the method is suitable for expression regulation of pleiotropic genes. Different levels of expression down-regulation effects can be obtained by accurately mutating the basic groups in the region, so that the similar dose control on the gene expression level is realized.

The present application provides a method for producing circular RNA, the method comprising transcribing a vector to form a precursor RNA, wherein the vector contains the following elements operably linked to each other and arranged in the following order: a) a 5' element, b) a 3' Group I self-splicing intron fragment comprising a 3' splice site dinucleotide, c) an element that does not contain or contains an internal ribosome entry site (IRES) and a protein coding region, or an element that contains a non-coding region, d) a 5' Group I self-splicing intron fragment comprising a 5' splice site dinucleotide, and e) and a 3' element, wherein the 5' element and the 3' element form a stable structure with a Gibbs free energy (ΔG) of -190 kcal / mol to -9.0 kcal / mol, provided that the stable structure is not a duplex with at least 95% base pairs between the 5' element and the 3' element, and wherein the 3' Group I self-splicing intron fragment and the 5' Group I self-splicing intron fragment self-cleave and self-ligate to form a circular RNA molecule.

The application discloses a long-read RNA splicing consistency scoring and recognition method, comprising the following steps: (1) extracting candidate splicing reads from the SAM or BAM format alignment results of long-read RNA sequencing data; (2) analyzing the splicing site coordinates of the candidate splicing reads; (3) grouping the candidate splicing events; (4) calculating the splicing consistency score; (5) determining the candidate splicing events according to rules; and (6) machine learning enhanced determination. The application takes the spatial distribution characteristics of the splicing sites in multiple reads as the core, defines the splicing consistency score, and quantitatively represents the site stability of the candidate splicing events. On this basis, the read support, splicing sequence characteristics and machine learning enhanced determination module are further combined to realize the hierarchical recognition and probability evaluation of the candidate splicing events, so that the accuracy, robustness and discovery ability of potential new splicing events of the splicing site recognition in the long-read RNA sequencing data are improved.

The invention belongs to the field of bioinformatics and genomics, and discloses a method for rapidly and accurately predicting an intron splicing site of a target species. The method comprises the following steps: 1) obtaining a genome sequence and transcriptome sequencing data of a target species; 2) screening annotation genes and constructing a training set and a test set; 3) performing learning training on splicing site information by adopting a deep learning model architecture combining a convolutional neural network, a bidirectional long-short-term memory network and a self-attention structure; 4) performing bit-by-bit prediction on the input gene sequence by using the model obtained by training; and 5) judging the position of the splicing site according to the prediction score, and labeling and re-evaluating the abnormal or low-credibility splicing site. The calculation model provided by the invention can learn key sequence features at splicing sites, and can be extended to various species through transfer learning to realize high-precision introne boundary prediction. According to the method, the gene annotation efficiency can be remarkably improved, splicing abnormity caused by mutation can be accurately recognized, and the method has important application value for genomics research and gene function analysis and regulation.

The invention discloses snRNA for correcting protein C defects caused by exon-intron boundary mutation and application of the snRNA. Specific U1 small nuclear RNA (U1snRNA) and U7 small nuclear RNA (U7snRNA) are designed for protein C defects caused by abnormal mRNA splicing due to PROC gene exon-intron boundary mutation, so that recognition of 5'splicing sites is enhanced or recovered, and activated potential splicing sites are shielded. The small nuclear RNA is expressed through respective expression vectors, and corresponding U1snRNA or U7snRNA is correspondingly designed for each mutation. Cell experiment results show that the U1snRNA and the U7snRNA can remarkably recover correct splicing of abnormal PROC transcripts and improve the expression level and the functional activity of protein C. The invention provides a small nuclear RNA strategy based on RNA, which has potential gene therapy application value.

The present invention relates to a therapeutic oligonucleotide for use in treating a disease in a subject that is associated with or caused by a missense, nonsense or in-frame indel mutation in a gene of the genome of the subject, wherein the therapeutic oligonucleotide is capable of specifically hybridizing to the 5'-splice site, the 3'-splice site or an exonic splicing enhancer (ESE) of an out-of- frame exon in the gene with the missense, nonsense or in-frame indel mutation, thereby causing skipping of the out-of-frame exon.