90 results about "Stop codon" patented technology

The invention discloses a construction method and application of a mouse model for specifically tracing heart valve cells, and belongs to the technical field of animal model construction. The model construction method comprises the following steps: (1) constructing a Wid1-Cre hybrid mouse before inserting a Cre recombinase coding gene into a termination codon of a mouse Wid1 gene; and (2) hybridizing the Wid1-Cre hybrid mouse with the Rosa26-tdTomato homozygous report mouse, so as to obtain the double transgenic mouse which simultaneously carries the Wid1-Cre gene and the Rosa26-tdTomato gene. According to the invention, on the basis of the expression characteristic that Wif1 is only limited to a valve area in the heart, Wif1-Cre is constructed; according to the Rosa26-tdTomato mouse model, specific tracing of heart valve cells is achieved, and an important experimental tool is provided for mechanism research of heart valve diseases and development of therapeutic drugs.

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.

This vector for nucleic acid introduction contains a nucleotide sequence that encodes a Cas protein, a nucleotide sequence that encodes a guide RNA, and a nucleotide sequence of a nucleic acid to be introduced. The guide RNA targets an intron adjacent to the 5' side, with reference to a sense strand, of an exon that includes a stop codon. Also provided is a nucleic acid introduction method comprising a step for introducing a nucleic acid to be introduced through non-homologous end joining by targeting an intron that is adjacent to the 5' side, with reference to a sense strand, of an exon that includes a stop codon.

Provided is an efficient and precise plant gene knockout method. First, a sequence containing a stop codon cluster is inserted into a genome by means of dual pegRNA to obtain, by means of screening, a stop codon cluster sequence capable of efficiently and precisely knocking out a target gene. A prime editing system and multiple pegRNA programming can be used in combination with the method to enable various genomic modifications such as efficient and precise knockout of one or more target genes, base substitutions, and small fragment insertions, deletions, and substitutions with just one prime editing protein.

The invention discloses an mRNA sequence scoring method and related equipment. The method comprises the following steps: acquiring an mRNA sequence to be scored; extracting a key base sequence from the mRNA sequence, wherein the key base sequence comprises an initiation codon context and a termination codon context; inputting the key base sequence into a target scoring model, and processing to obtain a score of the mRNA sequence in the target index; wherein the target scoring model comprises a multi-modal RNA sequence coding module and a large language model, and the target scoring model is obtained based on unsupervised pre-training of transcription sample data and supervised fine tuning of a score label data set corresponding to the target index. According to the method, a non-coding region and a coding region key region (start / stop codon context) are integrated as input, and a large language model trained based on mass data is used for processing, so that various complex factors such as sequence context information and secondary structures can be comprehensively considered, and the method can be widely applied to the technical field of gene sequence analysis.

A bioengineering method which comprises introducing an inert nucleic acid cassette into a biological entity without introducing or modifying characteristics or traits in the biological entity. The method comprises receiving or providing a sample comprising the biological entity having a nucleic acid sequence; selecting an integration site in the nucleic acid sequence for inserting the inert nucleic acid cassette; designing the inert cassette with optimized primer sequences, optimized probe sequences, optimized stop codons and disrupted start codons, and inserting the inert nucleic acid cassette into the biological entity at the integration site; and validating that no characteristics have been added or modified in the biological entity.

The invention relates to the technical field of biomedicine, and particularly provides a mouse model capable of being inductively combined with epithelial cell specific ablation and application of the mouse model. The method comprises the following steps: inserting a '3xFLAG-P2A-CreERT2 'functional frame at a fixed point at a termination codon of a mouse Odam gene, so as to obtain an OdamCreERT2 mouse. The expression specificity of the Odam gene in binding epithelium is verified by a mouse obtained by copulating with a ROSA26mT / mG reporter gene mouse; mice obtained by mating with ROSA26DTA cell ablation mice reveal the indispensable function of binding epithelium in maintaining periodontal barrier and preventing bone resorption. The model retains endogenous Odam expression and functions, supports protein dynamic tracing and space-time specific gene manipulation, and is suitable for researching the effect of binding epithelium in periodontal barrier integrity and alveolar bone resorption prevention.

The invention discloses an influenza reporter virus carrying a fluorescent RNA aptamer and a construction method and application thereof, and the influenza reporter virus carrying the fluorescent RNA aptamer is obtained by introducing a termination codon TAA into the 328th nucleotide of an ORF region of an NS1 gene on the basis of a genome of an influenza A virus and terminating in advance, then introducing a terminating codon TAA into the 328th nucleotide of the ORF region of the NS1 gene of the influenza A virus; original nucleotides at the 328-480 sites are replaced by forward or reverse RNA aptamers, and the RNA aptamer is obtained. The influenza reporter virus carrying the fluorescent RNA aptamer constructed by the invention also has the characteristics of good virus replication ability, high RNA imaging sensitivity and good specificity on the basis of stable inheritance, can be used for real-time visual observation of virus RNA, and is used for research on virus replication cycle and molecular mechanism related to the virus RNA. Besides, the construction method of the influenza reporter virus carrying the fluorescent RNA aptamer can also be applied to construction of RNA fluorescent reporter viruses of other subtype influenza viruses such as H3N2 and H5N1, and an important tool is provided for real-time tracing of corresponding virus RNA.

Aspects of the disclosure relate to methods, uses, compositions, kits, and systems for editing an endogenous tRNA into a suppressor tRNA. Additional aspects relate to compositions comprising the prime editing machinery, pegRNAs, and / or complexes comprising the prime editor and pegRNA that are capable of editing and / or replacing an endogenous tRNA to yield a suppressor tRNA. Polynucleotides, cells, vectors, and complexes are also contemplated.

The invention relates to a nucleic acid molecule complex for pseudouridylation of a target RNA in a mammalian cell comprising a guide RNA (gRNA) that at one end is flanked by a first twister ribozyme and at its other end by a second twister ribozyme to generate a circular gRNA, wherein the nucleic acid molecule complex has the capability to generate circular gRNA which is characterized by a secondary structure comprising two pseudouridylation pockets that harbor antisense guide sequences. More specifically, the first twister ribozyme is a P3 twister ribozyme and the second twister ribozyme is a P1 twister ribozyme, wherein each twister ribozyme is separated from the gRNA by a linker. In a further aspect, the invention relates to a method for generating circular gRNA. The invention furthermore relates to a nucleic acid molecule complex as defined herein for use in the treatment of a genetic disease caused by a premature stop codon. It furthermore relates to a pharmaceutical composition, and a delivery vector to express the construct in mammalian cells to generate circular gRNA.

The invention relates to the field of bioengineering, in particular to a recombinant human herpes simplex virus as well as a construction method and application thereof. The recombinant human herpes simplex virus lacks US1, US2, US3, US4 and US5 virulence genes at the same time. The construction method comprises the following steps: constructing a donor plasmid, and constructing a cleavage plasmid; transfecting cells with the cleavage plasmids and the donor plasmids in proportion; and after the transfected cells are incubated, infecting with an HSV-1 virus, purifying, and verifying to obtain the recombinant virus. The donor plasmid constructed by the invention retains non-coding regions among US1, US2, US3, US4 and US5 genes, also retains initiation codons and termination codons of the US1, US2, US3, US4 and US5 genes, is added with a fluorescent tag sequence, and can knock out five virulence genes of HSV-1US1-US2-US3-US4-US5 at the same time. The neutralizing antibody titer result of the recombinant human herpes simplex virus is the same as that of a wild type, and a foundation is laid for vaccine preparation.

The present disclosure is concerned with piperidinedione compounds, pharmaceutical compositions comprising the compounds, and methods of treating disorders associated with the presence of a premature termination codon such as, for example, cystic fibrosis, Duchenne muscular dystrophy, aniridia, Becker muscular dystrophy, spinal muscular atrophy, Hurler syndrome, hemophilia, epidermolysis bullosa (e.g., dystrophic (DEB) form, junctional (JEB) form). Usher syndrome, and cancer, using the compounds. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

The application discloses a molecular marker of a new functional allelic variant Sbdth1 of a sorghum heading stage gene SbDTH1 and application of the molecular marker. The application provides regulation of a functional allelic variant in the sorghum heading stage gene SbDTH1, and the fourth base after a termination codon TGA is inserted with a transposon. The allelic variant can prolong the heading time of sorghum. A molecular marker for high-throughput marker molecular detection is developed according to the allelic variant. The molecular marker has advantages of flexibility, high efficiency, high throughput, low cost and the like. The molecular marker can be used for detection and screening of the allelic variant, used for molecular marker breeding, and used for breeding of new sorghum varieties with suitable heading stages. The application has important significance for expansion of regional planting of sorghum and improvement of yield.

Aspects of the disclosure relate to methods, compositions, and systems for editing a DNA sequence encoding an endogenous tRNA into a suppressor tRNA using base editing (e.g., to treat a disease caused by a premature termination codon or PTC). Additional aspects relate to compositions comprising a gRNA configured to bind to a DNA sequence encoding an endogenous tRNA. Other aspects relate to complexes comprising a base editor and a gRNA that are capable of editing an endogenous tRNA into a suppressor tRNA. In some aspects, the disclosure further relates to polynucleotides encoding one or more nucleic acid sequences encoding the gRNAs, vectors comprising the polynucleotides, and / or cells comprising the polynucleotides, complexes, gRNAs, and / or vectors disclosed herein. Additional aspects further relate to kits comprising any one of the compositions, complexes, gRNAs, polynucleotides, vectors, and / or cells disclosed herein.

The application discloses an mRNA sequence scoring method and related equipment, and the method comprises the following steps: acquiring an mRNA sequence to be scored; extracting a key base sequence from the mRNA sequence, wherein the key base sequence comprises a start codon context and a stop codon context; inputting the key base sequence into a target scoring model to obtain a score of the mRNA sequence on a target index; wherein the target scoring model comprises a multi-modal RNA sequence encoding module and a large language model, and the target scoring model is obtained based on unsupervised pre-training of transcription sample data and supervised fine-tuning of a score label data set corresponding to the target index. The application integrates non-coding regions and key regions of coding regions (start / stop codon contexts) as inputs, and processes them by using a large language model trained based on massive data, so that the application can comprehensively consider various complex factors such as sequence context information and secondary structure, and can be widely applied to the technical field of gene sequence analysis.

The invention belongs to the technical field of veterinary biological products, and relates to an mRNA freeze-dried vaccine of a porcine epidemic diarrhea virus as well as a preparation method and application of the mRNA freeze-dried vaccine. When the mRNA vaccine is prepared, firstly, the S gene sequences of the porcine epidemic diarrhea virus GIIa type and the porcine epidemic diarrhea virus GIIc type which are separated and stored in a laboratory are selected; the preparation method comprises the following steps: firstly, carrying out codon optimization, then respectively connecting with 5 'UTR, 3' UTR, a termination codon and a polyA tail nucleic acid sequence in series, connecting to a vector, carrying out in-vitro template transcription, carrying out in-vitro transcription, capping and purifying to obtain mRNA, and finally, encapsulating and freeze-drying to prepare the mRNA vaccine. The invention further provides application of the vaccine to immunization of pigs, cellular immunity and humoral immunity in the pigs can be stimulated, the immunogenicity and protective efficacy of the vaccine are superior to those of commercially available bivalent live vaccines, and the vaccine can be used for preventing porcine diarrhea caused by infection of currently epidemic PEDV GIIa and GIIc type strains.

The present disclosure includes bioorthogonal conjugation systems and methods of producing same. The systems include at least one ncAA that participates in an orthogonal click reaction owing to its reactive group. The system further includes at least one plasmid having a gene for one or more of: an aminoacyl-tRNA synthetase, a tRNA, and a nanobody. Each nanobody includes suppression stop codons that define locations for conjugation of a ncAA, such that reactive groups of the ncAAs are incorporated at each defined location of the nanobody. Locations are not restricted to N and C terminals of the peptide, facilitating nanobody functionality post-conjugation. Resulting nanobodies are modified with the reactive groups of the ncAA in one or more locations, such that multiple cargos can be conjugated to each nanobody. Additionally, nanobodies can be conjugated to other nanobodies having corresponding reactive groups to form assemblies, which may be used as therapeutic nanobody libraries.

To provide a method for rescuing a termination codon through genetic reassignment using ACE-tRNA.SOLUTION: In certain embodiments, the invention provides a modified transfer RNA (tRNA) comprising a T-arm, a D-arm, an anticodon arm, and an acceptor arm, wherein the T-arm comprises a T-stem having a nucleotide that interacts with elongation factor 1 alpha 1 (EF1 alpha). EF1 alpha recruits aminoacyl-tRNAs to the ribosome and protects the tRNA from being deacylated. Rational nucleotide substitutions result in tuned tRNA: EF1 α interactions that enhance tRNA delivery to the ribosome and protection from de-acylation.SELECTED DRAWING: None

The application of OsFabF gene in resistance to rice stripe virus, the application relates to the technical field of rice disease resistance breeding. The application uses a CRISPR / Cas9 gene editing system to design sgRNA targeting a specific functional region of an endogenous OsFabF gene in rice, to precisely edit the region, thereby obtaining a mutant with a functionally deficient OsFabF. The editing preferably results in a frame shift mutation or a premature stop codon, to obtain a truncated OsFabF protein mutant. Experiments show that the rice mutant plant obtained in this way exhibits significantly enhanced resistance to RSV infection, reduced virus accumulation and reduced disease symptoms. The application provides an effective gene target and breeding material for cultivating new rice varieties resistant to rice stripe virus.

This invention relates to a mouse model of Parkinson's disease and its applications. The construction method includes the following steps: 1) in mice... Snack An exogenous fragment containing the human SNCA G51D coding sequence was knocked into the region upstream of the gene stop codon via homologous recombination, causing the human SNCA G51D coding sequence to interact with the mouse endogenous gene. Snack The gene forms a bicistronic expression structure, and the coding sequence of the human SNCA G51D is shown in SEQ ID No:1; 2) Positive F0 generation mice are backcrossed with wild-type mice, and genotypes are identified and screened to obtain a stable SNCA G51D endogenous knock-in Parkinson's disease mouse model. The gene editing strategy of the Parkinson's disease mouse model preserves the mouse's... Snack Simultaneous endogenous expression of α-synuclein G51D in the gene can mimic two pathogenic forms, exhibiting high physiological relevance and genetic authenticity.

A self-circularized RNA structure of the present invention can be expressed in a DNA vector and, at the same time, form circRNA by being circularized through a self-targeting and splicing reaction, wherein the circRNA consists of only a gene of interest. The gene of interest includes an IRES region, an initiation codon and a termination codon, and thus enables the rapid expression of a peptide and a protein.

The present application relates to a kind of efficient salvia miltiorrhiza single base editing system and its application, belong to base editing technical field.The present application provides a kind of salvia miltiorrhiza single base editing system, the salvia miltiorrhiza single base editing system includes 35SEN complex promoter, sgRNA, SmRPS5A promoter and fusion protein;The fusion protein is SmABE8e fusion protein or SmAKBE fusion protein.According to the present application, efficient salvia miltiorrhiza single base editing system SmABE8e-03 and SmAKBE-03 are successfully constructed, can edit the base of salvia miltiorrhiza, and salvia miltiorrhiza single base editing system can edit the 5'UTR of SmMYB1 gene and the termination codon of SmKSL2 protein, by destroying uATG, ORF and protein structure, can improve the content of tanshinone and salvianolic acid in salvia miltiorrhiza under the condition of not affecting its growth trait.

This invention discloses a method for constructing a Brca2-p.Q462Ter genetically engineered mouse and its application. The Brca2 genetically engineered mouse provided by this invention carries a truncated mutation in its Brca2 gene. This mutation is located at exon 10 of the mouse Brca2-201 transcript, changing amino acid position 462 from glutamine Q (Gln) to the stop codon Ter, forming the Brca2-p.Q462Ter mutation. The Brca2-p.Q462Ter genetically engineered mouse is used to simulate the human BRCA2 gene truncated mutation p.Gln472Ter, c.1414C>T. Amino acid homology analysis shows that the mouse BRCA2 protein Q462 site is homologous to the human BRCA2 protein Q472 site; therefore, this mouse mutation site can be used to simulate the corresponding truncated mutation effect in human BRCA2. This genetically engineered mouse can simulate, at the whole animal level, the abnormal DNA damage repair, impaired homologous recombination repair function, decreased genome stability, and susceptibility to related diseases caused by BRCA2 truncated mutations, thus providing a reliable tool for research on BRCA2-related pathogenesis, developmental effects, drug screening, and efficacy evaluation.

The invention belongs to the field of biosynthesis, and particularly relates to a CHO K1 (ECACC) cell strain with completely inactivated glutamine synthetase (Glul) gene functions as well as a preparation method and application of the CHO K1 (ECACC) cell strain. According to the invention, a base editor CBE4max-SpRY is adopted to target a Glul gene coding region, and a specific codon is efficiently and accurately mutated into a termination codon (TAG / TAA / TGA) through cytosine base directional conversion (C.G-T.A) guided by guide RNA (gRNA) under the condition of not introducing DNA double-strand breakage (DSB), so that complete knockout of the Glul gene is realized. The yield of stably transfected foreign protein of the obtained cell strain is obviously higher than that of commercial CHOZN and ECACC CHO K1 cell lines, and the GS06-10 clone expression quantity of a targeted exon 6 is improved by 3 times compared with that of an ECACC CHO K1 wild type.

In certain embodiments, the present invention provides a modified transfer RNA (tRNA) comprising a T-arm, a D-arm, an anticodon-arm and an acceptor arm, wherein the T-arm comprises nucleotides that interact with the elongation factor 1 alpha protein, and methods of use thereof. In certain embodiments, the present invention provides a modified transfer RNA (tRNA) comprising a T-arm, a D-arm, an anticodon-arm and an acceptor arm, (a) wherein the anticodon-arm comprises a tri-nucleotide anticodon, wherein the anticodon is 5′-UCA-3′ and recognizes TGA stop codons, and wherein the acceptor arm is operably linked to a arginine, tryptophan or glycine; (b) wherein the anticodon-arm comprises a tri-nucleotide anticodon, wherein the anticodon is 5′-UUA-3′ and recognizes TAA stop codons, and wherein the acceptor arm is operably linked to a glutamine or, glutamate; or (c) wherein the anticodon-arm comprises a tri-nucleotide anticodon, wherein the anticodon is 5′-CUA-3′ and recognizes TAG stop codons, and wherein the acceptor arm is operably linked to a tryptophan, glutamate or glutamine.

The present disclosure is concerned with substituted uracil compounds, pharmaceutical compositions comprising the compounds, and methods of treating disorders associated with the presence of a premature termination codon such as, for example, cystic fibrosis, Duchenne muscular dystrophy, aniridia, Becker muscular dystrophy, spinal muscular atrophy, Hurler syndrome, hemophilia, epidermolysis bullosa (e.g., dystrophic (DEB) form, junctional (JEB) form), Usher syndrome, and cancer, using the compounds. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

The invention belongs to the technical field of gene editing, and particularly discloses a Crimson fluorescent protein reporting system and application thereof in gene editing efficiency multi-scene evaluation. According to the Crimson fluorescent protein reporting system, an equidirectional repetitive sequence which can be targeted by sgAlb and contains a termination codon is introduced into a Crimson gene sequence, a frame-shift mutant fluorescent reporting module is successfully constructed, and the module can recover fluorescent expression through an SSA (homologous sequence annealing repair) repair mechanism after being cut in a Cas9 / sgAlb system. The developed Crimson report cell system can be used for accurately evaluating the stability and editing efficiency of different sgRNA skeletons; the method is suitable for optimizing a virus transduction system and screening a guiding aid for efficient gene delivery; plasmid pollution in a virus vector system can be rapidly detected, and accurate distinguishing of a functional RNP compound and residual plasmid DNA is realized by comparing the proportion of Crimson positive cells before and after DNase I treatment and heat treatment; and small molecular compounds for promoting homologous directional repair can be efficiently screened.

Disclosed are compositions and methods for treating diseases of the mammalian eye, and in particular, complications of the retina associated with Usher syndrome 1B (USH1B). Further disclosed are compositions and methods for treating diseases of the mammalian inner ear, and in particular, complications of ear hair cells associated with Usher syndrome 1B (USH1B). The disclosure provides improved AAV-based, dual vector systems that facilitate the expression of full-length proteins whose coding sequences exceed that of the polynucleotide packaging capacity of an individual AAV vector. Described herein are modified hybrid dual vector systems that shift the coding sequence for the MYO7A tail domain from the front-half vector to the back-half vector by altering the split point (e.g., from between exons 23 and 24, to between exons 21 and 22), in order to eliminate the production of truncated MYO7A protein. Further described herein are improved, codon-modified hybrid and overlap vector systems in which putative stop codons and residual sequences in non-coding sequences are removed.

The present invention relates to a purine derivative for use in the treatment of a disease due to a nonsense mutation in a gene leading to the premature introduction of a UGA stop codon.