137 results about "Stop codon" patented technology

The invention discloses a quinoa CqARF2 gene mutant as well as an identification method and application thereof. Chenopodium quinoa seeds are mutated on a large scale by utilizing a chemical mutagenic agent EMS, a CqARF2 gene function deletion mutant arf2 is adopted as a quinoa mutant, G at the 1050th site of the CqARF2 gene of the mutant is mutated into A, and amino acid at the 350th site is mutated into a termination codon, so that the translation process of CqARF2 protein is terminated in advance. The chenopodium quinoa CqARF2 gene function deletion mutant arf2 shows remarkable agronomic character improvement, seeds are large and full, compared with a wild type, the hundred-grain weight of the mutant seeds is increased by about 40%, the seed diameter is increased by about 10%, the seed thickness is increased by about 30%, and a certain foundation is laid for breeding new high-yield and high-quality chenopodium quinoa varieties.

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 present application provides methods, compositions, and systems for decoding a pseudouridine-modified (psi-modified) codon. In some aspects, the present application provides methods for improving the translation of a target RNA in a host cell, the target RNA comprising a psi-modified codon wherein the method comprises: introducing a near homologous transport RNA (nctRNA) of the psi-modified codon or a nucleic acid encoding the nctRNA into the host cell; and wherein the psi-modified codon is decoded to an amino acid, thereby improving translation of the target RNA. In some cases, the codon of the psi modification is a premature termination codon (PTC).

The present subject matter relates to recombinant lentiviral transfer plasmids for producing lentiviral vectors with increased vector titer and performance. These transfer plasmids are constructed so that a central polypurine tract (cPPT) is centered between the lentiviral LTRs and include a 6x stop codon sequence block downstream of the 3' LTR. When the transfer plasmids comprise a globin expression cassette, the lentiviral vectors produced therefrom are useful for treating hemoglobinopathies by gene therapy. Transduced cells, pharmaceutical compositions and methods of treating or ameliorating hemoglobinopathies with these lentiviral vectors are also provided. Additional transfer plasmids, cells, and compositions are provided that are useful for delivering any gene of interest to a cell, as well as plasmids for production of lentiviral vectors for treating other erythroid-specific diseases or disorders such as anemia and cancer.

The invention provides an in-vitro cell-free protein synthesis system for inserting non-natural amino acid. The reaction system comprises: (1) a yeast cell extract; (2) non-natural amino acids; (3) an exogenous orthogonal aminoacyl tRNA synthetase / orthogonal tRNA pair; (4) a template containing a target protein gene sequence, wherein a codon for coding at least one amino acid in the target protein gene sequence is mutated into a termination codon; by adopting the reaction system, the insertion efficiency of the non-natural amino acid and the expression quantity of the non-natural amino acid protein can be improved.

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 present disclosure provides compositions and methods for altering auxin accumulation in corn or maize plants. Methods and compositions are also provided for altering the expression of genes related to auxin efflux through editing or mutagenesis of a brachytic2 (br2) gene to introduce a premature stop codon or a deletion into the gene such that a truncated Br2 protein encoded by the mutant allele of the br2 gene, which may be a dominant or semi-dominant allele, has at least part of a transmembrane domain without a nucleotide binding domain or motif. Modified plant, plant parts and cells having such a mutant allele with reduced or altered expression or activity of a br2 gene product can have improved characteristics, such as reduced plant height and increased lodging resistance, but without off-types in the plant.

The invention relates to a corn ZmmtRF2a gene mutation marker, an identification primer and application of the corn ZmmtRF2a gene mutation marker in regulation and control of grain development. A natural grain mutant crk5 found in a corn field breeding line selection process is utilized, a target gene ZmmtRF2a causing crk5 grain mutation is obtained through phenotypic analysis, linkage analysis and map-based cloning technologies, the gene encodes a peptide chain release factor 2a for mitochondrial localization, the peptide chain release factor 2a affects the mitochondrial function, and the gene can be used for inducing the mutation of the crk5 grain. The development of corn embryos and endosperm and the accumulation of grain storage substances are regulated and controlled. In crk5, ZmmtRF2a lacks 5th to 7th exons, the encoded truncated protein does not have GGQ and SPF motifs, the motifs are crucial to release activity of RF, mitochondrial binding and termination codon recognition, technical support is provided for utilization of the gene, and a new way is opened up.

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.

The present invention discloses a method for constructing acid-resistant engineered algae, belonging to the fields of biology and molecular biology. The specific method is as follows: Using Phatr3_J33543 with the sequence structure shown in SEQ ID No.1, Phatr3_J50516 with the sequence structure shown in SEQ ID No.2, or Phatr3_Jdraft1806 with the sequence structure shown in SEQ ID No.3 as the target gene, removing the stop codon of the target gene, and connecting the fluorescent protein gene without the start codon through the DNA sequence encoding glycine to obtain a recombinant gene; inserting the recombinant gene into the multiple cloning site of the pPhaNR plasmid to obtain a vector plasmid; introducing the vector plasmid into diatom cells by electroporation and then inoculating them onto the ESAW solid selection medium containing bleomycin, and growing brown colonies; selecting the colonies with fluorescent protein signals in the colonies, which are the acid-resistant engineered algae. Compared with the wild algal species, the acid-resistant ability of the engineered algae obtained by the present invention is significantly improved. The present invention has important reference significance for the cultivation of acid-resistant diatoms.

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 present invention provides a chimeric DNA molecule including: (a) a first nucleic acid sequence encoding a polypeptide of interest, wherein a 3' end the first nucleic includes a stop codon; and (b) a second nucleic acid sequence encoding an endogenous polypeptide of a target cell, wherein the first nucleic acid sequence is located upstream to the second nucleic acid sequence in the chimeric DNA molecule, and wherein the first and second nucleic acid sequences are operably linked. Further provided is a method for increasing expression of a polypeptide of interest in a target cell, including culturing a cell including the chimeric DNA molecule, such that a polypeptide of interest encoded by the first nucleic acid sequence of the chimeric DNA molecule is expressed.

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 invention provides a preparation method and application of a CBD-AL fusion recombinant protein, a variant Z region obtained by site-directed mutagenesis of a B region of SPA is connected in series with a B structural domain of a protein L, codon optimization is carried out, the front end of CBD is introduced into a BamHI restriction enzyme cutting site, a cysteine sequence, a termination codon TAA and an EcoRI restriction enzyme cutting site are added to the tail end of the B region of the recombinant protein L, and the CBD-AL fusion recombinant protein is obtained. BamHI and EcoRI are subjected to double enzyme digestion and then are connected to a PET28a vector subjected to double enzyme digestion, a recombinant protein CBD-AL gene expression vector of a structural domain of strongest binding IgG of protein A and protein L connected with CBD is constructed, the recombinant protein CBD-AL gene expression vector is transferred into an expression strain for amplification culture, an inducer is added for induction, bacterial sludge is collected, purification is performed by cellulose affinity chromatography, and the recombinant protein CBD-AL gene expression vector is obtained. A binding protein which has a cellulose binding function and can bind all types of immunoglobulins and subtypes thereof is prepared.

The invention discloses a molecular marker closely linked with the PVY resistance of a sun-cured tobacco variety Pan County large willow leaf and application of the molecular marker, the molecular marker is located at the 267th-268th sites of an eIF4E1 gene cDNA coding region, double-base deletion occurs, frame shift mutation is caused, and an early termination codon is generated in a second exon. When the molecular marker of the tobacco PVY recessive disease-resistant gene is used for assisted selective breeding, the acceleration of the breeding process is promoted.

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 invention provides a preparation method and application of MBP-GL fusion recombinant protein. A C3 region of SPG and a B structural domain of protein L are connected in series, codon optimization is performed on a gene sequence, the front end of MBP is introduced into a BamHI restriction enzyme cutting site, a cysteine sequence, a termination codon TAA and an EcoRI restriction enzyme cutting site consistent with an expression vector are added to the tail end of the B region of the protein L, and the MBP-GL fusion recombinant protein is obtained. BamHI and EcoRI are subjected to double enzyme digestion and then are connected to a carrier subjected to the same double enzyme digestion, a recombinant protein MBP-GL gene expression carrier of a structural domain of strongest binding IgG of protein G and protein L connected with MBP is constructed, the carrier is subjected to transformation, induced expression and purification, and the recombinant protein MBP-GL gene expression carrier which not only has glucan ligand specific binding capacity, but also can be used for preparing the recombinant protein MBP-GL gene expression carrier. And the recombinant protein can be combined with all antibodies, subtypes and antibody fragments with high load.

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.

The present invention relates to a method of integrating an exogenous DNA sequence into a genome of a cell comprising contacting the cell with a) a donor nucleic acid comprising: —at least one STOP codon and a translation initiation sequence (TIS) or —a ribosomal skipping sequence, and —said exogenous DNA sequence wherein said donor nucleic acid is flanked at 5′ and 3′ by inverted targeting sequences; b) a complementary strand oligonucleotide homologous to the targeting sequence and c) a nuclease that recognizes the targeting sequence.

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 discloses a grass carp reovirus type II mRNA (messenger Ribonucleic Acid) vaccine as well as a preparation method and application thereof, and belongs to the field of vaccines. According to the technical scheme, the grass carp reovirus type II mRNA vaccine comprises a modified mRNA chain, the modified mRNA chain sequence comprises a 5 'UTR sequence, a Kozak sequence, a grass carp immunoglobulin mu heavy chain signal peptide coding sequence, a GCRV II outer capsid protein VP35 coding sequence, three termination codons, a 3' UTR sequence and a polyA nucleotide sequence. The GCRV II mRNA vaccine is applied to enhancing the GCRV infection resistance of a fish body, the death rate is reduced, and the blank of the current grass carp reovirus II mRNA vaccine is filled.

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 invention belongs to the technical field of gene engineering, and particularly relates to a neural crest stem cell tracing system based on P75NTR-T2A-eGFP and application. The invention focuses on construction and application of a tracing system for differentiating pluripotent stem cells into neural crest stem cells in vitro. By constructing a Crispr / Cas9 vector, a termination codon region of a P75NTR gene expression cassette is precisely targeted. Meanwhile, a gene knock-in vector containing an (LHA) left homologous arm, T2A-eGFP-right homologous arm (RHA) is constructed. The two vectors are introduced into pluripotent stem cells (hPSCs) by means of electric shock transfection. Through the operation, the P75NTR-T2A-eGFP gene knock-in pluripotent stem cell line is successfully obtained, a foundation is laid for building a follow-up tracing system, and the P75NTR-T2A-eGFP gene knock-in pluripotent stem cell line has important application value in the field of neural crest stem cells.