59 results about "A-site" patented technology

The invention belongs to the technical field of genes, and discloses a method for efficiently expressing foreign protein based on an NC048599.1 site in a CHO cell genome. A site for stably expressing a protein in a CHO cell genome is located in a range of 69195000 to 69199000 of a CHO cell gene NC048599.1 with a sequence as shown in SEQ ID NO: 1 (Sequence Identifier Number 1), and the sequence of the CHO cell gene NC048599.1 is shown in SEQ ID NO: 1. Different protein genes are introduced at fixed positions in a CHO cell genome, and stable expression is carried out.

Provided herein are fusion proteins and related methods and systems for increasing the efficiency of genome editing using site-specific nuclease enzymes. These fusion proteins, systems, and methods may selectively increase a desired editing outcome (e.g., insertion of a donor polynucleotide sequence). Also provided are various useful compositions for producing and using these fusion proteins and practicing these methods.

A homologous recombination method of excising a specific DNA sequence from a DNA molecule. The method includes providing the DNA molecule including a pair of target sequences that are a target of a site-specific recombination enzyme and a pair of homologous sequences that are DNA sequences sharing homology with each other. The pair of target sequences are oriented in different directions from each other and arranged on opposite sides of the specific DNA sequence. The pair of homologous sequences are arranged on opposite sides of the specific DNA sequence at positions further outside of the pair of target sequences. The method further includes inducing homologous recombination between the pair of homologous sequences by action of the site-specific recombination enzyme on the DNA molecule so that the specific DNA sequence and the pair of target sequences are excised from the DNA molecule.

The invention relates to the field of biology, and discloses a double-antibody connection probe for detecting lactylated PGK1, a preparation method and application, the double-antibody connection probe comprises an antibody A, an antibody B and complementary oligonucleotides respectively coupled with the antibody A and the antibody B; the antibody A is a polypeptide which is subjected to K139-lactic acid modification and is used for recognizing a K139la site of the PGK1; the antibody B is a polypeptide which is used for identifying more than or equal to 50 amino acids away from the K139 site in the PGK1 and has the length of 12-15 amino acids; the double-antibody connection probe obtained by the invention realizes the ultra-sensitive detection of the targeted protein.

A method of altering a eukaryotic cell is provided including transfecting the eukaryotic cell with a nucleic acid encoding RNA complementary to genomic DNA of the eukaryotic cell, transfecting the eukaryotic cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site specific manner, wherein the cell expresses the RNA and the enzyme, the RNA binds to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site specific manner.

There is provided a method of altering a eukaryotic cell comprising transfecting the eukaryotic cell with a nucleic acid encoding an RNA complementary to genomic DNA of the eukaryotic cell, transfecting the eukaryotic cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site-specific manner wherein the cell expresses the RNA and the enzyme, and modifying the eukaryotic cell with the nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site-specific manner. The RNA binds to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site-specific manner.

Methods and compositions are provided for driving expression of a coding sequence that has been integrated within the genome of a cell. A donor polynucleotide is integrated within the genome so that a non-functional promoter is located downstream of a genomic target sequence. This genomic target sequence can be bound by a site-specific promoter activation tool, wherein the site-specific promoter activation tool drives robust expression of a coding sequence that is operably linked to the non-functional promoter.

The invention provides a site-specific mutagenesis vector and a use method thereof, and relates to the field of gene engineering in the biotechnological pharmaceutical industry, the vector contains BbsI restriction enzyme cutting sites except for a polyclone region, and site-specific mutagenesis comprises the steps of carrying out base mutation on the BbsI restriction enzyme cutting sites, removing the BbsI sites and keeping amino acid unchanged. According to the invention, a Golden gate method is adopted to construct the multi-copy vector, and a plurality of copies are directly assembled on a system in one step, so that the time is saved, and the tedious steps of multiple times of enzyme digestion and connection are omitted. According to the invention, a multi-copy vector is constructed in vitro, the copy number of the antibacterial peptide is increased, simultaneous expression of each unit is realized, and the expression quantity is improved. Meanwhile, the antibacterial peptide genes are connected in series by adopting a serial connection technology of the antibacterial peptide genes, so that the expression quantity of the antibacterial peptide is improved on the DNA level.

A DNA editing method for inserting a desired nucleotide sequence into a target site of a target DNA in a cell or removing the target site includes an introduction step of introducing a vector and a site-specific nuclease system into the cell and bringing the vector and the site-specific nuclease system into contact with the target DNA, the vector comprises a first promoter P1 and structure (1): 5 '-M1-Hv1-D-Hv2-M2-3', M1 represents a 5 '-side fragment of a nucleotide sequence encoding a selection marker gene and is operably linked to P1, Hv1 represents a nucleotide sequence homologous to a first nucleotide sequence Ht1 on the 5' side of a target site of the target DNA and D-Hv2-M2-3 'represents a nucleotide sequence homologous to a second nucleotide sequence Ht2 on the 5' side of the target site of the target DNA and D-Hv2-M2-3 'is operably linked to P1. D represents the desired nucleotide sequence but may not exist, Hv2 represents a nucleotide sequence homologous to a second nucleotide sequence Ht2 on the 3'side of the target site of the target DNA, and M2 represents the remaining 3 '-side fragment of the nucleotide sequence encoding the selection marker gene; the DNA editing method comprises a cutting process, an editing process and a selecting process, and the cutting process is carried out through the site-specific nuclease system. And a step in which a fragment represented by structure (2): 5 '-Hv1-D-Hv2-3' and a fragment represented by structure (3): 5 '-M2-P1-M1-3' are generated from the vector, and the target site or the surroundings thereof is cleaved in the target DNA, in which the target DNA is bound to structure (2) depending on the homology of Ht1 and Hv1 and bound to structure (2) depending on the homology of Ht2 and Hv2, and the fragment represented by structure (3): 5 '-Hv1-D-Hv2-3' is cleaved in the target DNA depending on the homology of Ht2 and Hv2. And a selection step for obtaining a selection vector containing a selection marker gene that binds to the 3'terminal of M1 and the 5 'terminal of M2 in the fragment represented by structure (3) and is operably linked to P1, and that can function, whereby the desired nucleotide sequence D is inserted into the target site or the target site is removed, and a selection step for obtaining a selection vector that contains a selection marker gene that binds to the 3' terminal of M1 and the 5 'terminal of M2 in the fragment represented by structure (3).

The invention relates to the technical field of bioengineering and provides a method for improving the specificity and affinity of an aptamer. The method includes: S1, screening a target of an aptamer from a compound information database by virtual computing; S2, verifying the screening result in Step S1 through experiments; S3, performing virtual saturation mutation on a site of the aptamer, and screening out a mutation site of the aptamer; S4, performing base substitution to the mutation site of the aptamer; and S5, detecting the binding parameter of the aptamer after base substitution with the target screened in Step S1, and selecting an aptamer with improved specificity and affinity after base substitution. An efficient molecular design-guided method is developed by computer rational calculation, to improve the specificity and binding affinity of the aptamer by directional modification. The present invention is of great significance for the practical application of aptamers.

The application discloses a pig HBB gene site-directed modification system and application. The application provides a system for site-directed modification of a pig HBB gene, the first carrier, the second carrier and the third carrier contained in the system can express gene editing proteins and sgRNA, three target sites of the HBB gene are effectively cleaved, a site-directed modification fragment of a donor DNA is used to replace a to-be-site-directed modification fragment of the target site, and accurate deletion of bases from 176 to 179 of a CDS sequence of the HBB gene is realized. 41‑42(‑CTTT) The model accurately simulates the most common beta thalassemia mutation genotype beta of Chinese population, provides a precise model for analyzing the pathogenesis of beta thalassemia of Chinese population and developing a new treatment method.

The present invention provides a chimeric polyketide synthase (PKS) or polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) and uses thereof, wherein the PKS or PKS-NRPS comprises two or more recombinant units (RUs) linked to each other, wherein each RU comprises one or two or more contiguous domains derived from a parent PKS or PKS-NRPS, and the junction of at least one RU is defined by a recombination site selected from a site at the LPTY (A / P) FQ (H / R) xRYWL motif located in the AT post- linker (RYWL motif site) and a site at the RL motif of ACP helix 0 (RL motif site); wherein the two or more RUs are not naturally linked to each other.

A protein-glycan conjugate having multiple payloads. By means of a site-specific glycan conjugation technique, different types of payloads are conjugated to specific positions of a protein-glycan chain, so as to obtain a conjugate having multiple payloads. The conjugate exhibits good biological effects.

Disclosed herein is a method for training a framework comprising one or more models for site-specific ribonucleic acid (RNA) modification detection on an RNA sequence of interest, comprising receiving a training dataset of sequences, each sequence being either an unmodified RNA sequence or a modified RNA sequence and, for each sequence, annotations indicating whether the respective sequence contains a modification at one or more sites; extracting a sequence context read j from each sequence, and extracting, for each site of interest i in read j, i being a site at which it is desired to detect a modification, a site-specific annotation from the annotations, the site-specific annotation indicating whether a specific modification m occurs at site i; training at least one said model to determine a probability of modification m at site i, using a plurality of k-mers, extracted from each read j, including one or more k-mers from before site i, at site i, and after site i; updating the at least one model based on a loss between the probability and the site-specific annotation; and outputting the framework, after a predefined number of epochs for training has been completed.

The application discloses a rice OsRDR5 gene and a coding protein, a site-directed editing system, application and an application method, a nucleotide sequence of the OsRDR5 gene is shown as SEQ ID NO. 1; and an amino acid sequence of the coding protein of the rice OsRDR5 gene is shown as SEQ ID NO. 2. The gene has a Coa3_cc domain and belongs to a cytochrome c oxidase assembly factor 3 gene family. Knockout mutants of the OsRDR5 gene show a higher deep root ratio, and simultaneously change the gravitropism of rice root systems. The rice OsRDR5 gene of the application regulates the drought avoidance of rice, the knockout mutants have a higher deep root ratio, and can be applied in breeding of new water-saving and drought-resistant rice varieties, so that the drought resistance of rice is improved.

A non-naturally occurring peptide linker, comprising a site-specific conjugation motif of GGX1X2Q, which allows for site-specific conjugation mediated by a transglutaminase; wherein each of X1 and X2 independently represents any naturally-occurring amino acid or one of X1 and X2 is absent; and wherein the non-naturally occurring peptide has a length of 9-20 amino acids. Also provided herein are fusion polypeptides such as tandem single-domain multi-specific antibodies containing such peptide linkers.

A method of altering a eukaryotic cell is provided including transfecting the eukaryotic cell with a nucleic acid encoding RNA complementary to genomic DNA of the eukaryotic cell, transfecting the eukaryotic cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site specific manner, wherein the cell expresses the RNA and the enzyme, the RNA binds to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site specific manner.

A site-specifically-modified engineered tRNA and the use thereof. Said modification refers to replacement, with one or more modified nucleotides, of one or more native unmodified nucleotides selected from sites 4, 9, 13, 14, 17, 18, 27, 28, 32, 34, 35, 36, 37, 38, 39, 49, 50, 54, 55, 58 and 72 of a tRNA molecule. Compared with unmodified tRNAs, the site-specifically-modified tRNA has higher structural stability and / or higher nuclease degradation resistance, the aminoacylation efficiency thereof is not remarkably reduced, and immunogenicity thereof is not remarkably increased. The modified tRNA can be used for eliminating premature translation terminations caused by nonsense mutations, and thus has great application potentials in treating nonsense mutation-related genetic diseases, tumors, etc. The modification of the engineered tRNA also provides valuable insights for drug development of tRNAs and thus can guide the modification design of native tRNAs.

This invention discloses a method based on SOD The primers and methods for gene screening of heat-resistant scallop varieties were developed using scallop cDNA and DNA as templates. PCR amplification was performed using upstream and downstream primers (SEQ ID No. 3 and SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6) to obtain samples containing scallop cDNA and DNA. SOD Gene sequence fragments from the exon and promoter regions were used as molecular markers. The genotypes were CT at the c.229 C>T site, CA at the c.284 C>A site, and CA at the c.348 C>A site in the exon region, and CA at the g.1124 A>C site and AT at the g.1359 T>A site in the promoter region. This method screened for high-temperature resistant scallop varieties, demonstrating advantages such as genetic stability, accuracy, and efficiency.

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

Provided are directed to compositions, methods, and systems for epigenetic editing of antigen presentation mechanism (APM) -related genes in a cancer cell. In some embodiments, provided are directed to methods of modulating methylation at a methylation site in an APM-related gene in a cancer cell. In some embodiments, the site-specific demethylation systems comprise a fusion protein or a nucleic acid encoding the fusion protein, wherein the fusion protein comprises a site-specific DNA binding domain and an effector domain having demethylation activity. In some embodiments, provided are directed to such methods, wherein recruitment of the site-specific demethylation system to a target site results in demethylation at the methylation site. Also provided herein are nucleic acids encoding the polypeptides described herein, systems comprising additional components useful for editing, such as gRNA, methods of treating cancer, and kits directed to practicing the methods described herein.

The present disclosure relates to a novel screening method for screening a plurality of pGREs in parallel and nucleotide constructs for use in the method. The construction body comprises a nucleotide block, the sequence of the nucleotide block is PRIMREG1-pGREE-PRIMREG2-GENEE or GENE-PRIMREG1-pGREE-PRIMREG2, and the PRIMREG1 is a primer recognition sequence 1 or an FLEx box containing the primer recognition sequence 1. PGRE is a presumed gene regulatory element, PRIMREG2 is a primer recognition sequence 2 or a FLEx cassette comprising a primer recognition sequence 2, GENE comprises nucleotide blocks REPSEQ, IRES and SSR, where REPSEQ is a reporter gene and is present or absent, IRES is an internal ribosome entry site or a 2A self-cleaving peptide, and SSR is a site-specific recombinase gene. At least one of the PRIMREG1 and the PRIMREG2 is a FLEx cassette comprising a primer recognition sequence.

The invention provides a sequencing chip and a sequencing method. The chip comprises: a substrate; the primer group is fixed on the surface of the substrate, the primer group comprises a first primer, the first primer is provided with a first cleavable site, and the first cleavable site is a site enabling the first primer to be cleaved under a first cleavage condition; the second primer is provided with a second cleavable site, and the second cleavable site is a site enabling the second primer to be cleaved under a second cleavage condition; the third primer does not contain the first and second cleavable sites, the second cleavable site is not cleaved under the first cleavable condition, the first cleavable site is not cleaved under the second cleavable condition, and the second and third primers have 80% or more of the same sequence. On the basis of the chip, a sequencing library cluster of a second-chain nucleic acid molecule and a first-chain nucleic acid molecule chain can be obtained through amplification, links such as terminal repair and second-chain nucleic acid molecule generation are not needed, the sequencing time and reagent cost are greatly reduced while the subsequent sequencing quality is guaranteed, and the sequencing efficiency is improved. The performance consistency of the second nucleic acid molecular chain and the first nucleic acid molecular chain is better.

The application provides a site-directed mutant Arabidopsis cryptochrome gene and its coded protein and application, compared with the prior art, the application obtains the sequence of the target gene CRY2 and connects with a pJL-blue plasmid, constructs a recombinant plasmid pJL-blue-CRY2; designs a mutant primer, and uses the recombinant plasmid to carry out site-directed mutation, constructs a mutant plasmid pJL-blue-D392N. Then the mutant plasmid is mixed with an expression plasmid in a reaction system, under the action of an exogenous LR recombinase, the target gene is recombined to the expression plasmid vector PFK272, and the mutant expression plasmid PFK272-D392N is transformed into Arabidopsis plants, and a transgenic Arabidopsis plant is constructed. Compared with the prior art, the transgenic plant D392N expressed by the application can accelerate the flowering of Arabidopsis, and the efficiency is about 25%. On the other hand, the number of rosette leaves will also be less.

The present disclosure relates to methods for increasing DNA methylation of a site in a genomic region comprising a target gene, e.g., using an expression repressor comprising a DNA targeting moiety that binds to a target sequence in the region and an effector domain (e.g., a DNA methyltransferase) that methylates DNA, or a nucleic acid encoding the expression repressor. Also disclosed are systems comprising two or more expression repressors. Compositions may be used, for example, to reduce expression of the target gene in a cell or subject, for example, to treat a condition associated with expression of the target gene.

Monocot plants heterozygous for centromeric histone 3 (CenH3) and optionally expressing gene editing constructs, for use in inducing haploids of a monocot target plant and optionally pass-through gene editing are provided. The monocot haploid inducer plants are typically composed of diploid plant cells having only one allele encoding a functional CENH3 protein. The diploid plant cells can also include, for example, one CenH3 allele encoding non-functional CENH3 protein. In some embodiments, the allele encoding non-functional CENH3 protein is a frameshift mutation, protein null allele, an RNA null allele, or a combination thereof. The monocot haploid inducer plant can also include gene editing machinery, such as a site-directed nuclease and optionally a guide RNA stably expressed by cells of the monocot plant. Methods of inducing formation of a target haploid monocot plant while optionally simultaneously modifying the target monocot plant's genome are also provided.

Provided is a method for preparing long-chain RNA, comprising a synthesis step, an annealing step, and a ligation step. The method for preparing long-chain RNA can realize chemical modification of any precise site in long-chain RNA through design of a first-strand RNA fragment and a second-strand nucleic acid fragment, so that the long-chain RNA has improved stability and improved immunogenicity and the like. The method can obtain a double-stranded assembly formed by complementation of a continuous single-strand RNA and a fragmented single-strand nucleic acid chain, and the double-stranded assembly only needs to be denatured to obtain a single-strand long-chain RNA, so that the synthesis step of the long-chain RNA is effectively simplified, the synthesis efficiency is improved, and the method is suitable for industrialized large-scale preparation.

Disclosed herein are fusion protein, comprising (a) at least one X1 domain comprising a half-life extension compound, including but not limited to a half-life extension polypeptide; (b) at least one X2 domain comprising an anionic block; (c) at least one X3 domain comprising a linker susceptible to cleavage at a site of disease, including but not limited to a microbial infection site or tumor site; and (d) at least one X4 domain comprising a therapeutic peptide; wherein the at least one X1, X2, X3, and X4 domains are covalently linked, and each X4 domain is linked to an X3 domain without an intervening X1 or X2 domain; compositions containing such fusion proteins, and methods for their use.

The invention relates to a method for tracing mononuclear macrophages. Comprising the following steps: placing a coding sequence of a fusion protein formed by connecting a site-specific recombinase and a ligand binding domain which can be combined with an exogenous ligand and regulate and control the activity of the recombinase after combination under the control of a regulation and control element of a C3aR1 gene, so that the expression of the fusion protein is limited in a cell with a C3aR1 expression history; setting an excidable blocking sequence corresponding to the recombinase recognition site in a genome of the recombinase recognition site, so that the blocking sequence inhibits the expression of a signal output molecule when the recombinase recognition site is not recombined; after the exogenous ligand is given, the recombinase is activated by regulating and controlling the conformational change of the ligand binding domain in the fusion protein, so that the recombinase is cut off the resectable blocking sequence, and the inhibition on the signal output molecule is relieved; and the signal output molecule for tracing is irreversibly expressed in the mononuclear macrophage with the C3aR1 expression history.

The present invention relates generally to the field of RNA splicing. In particular, the invention relates to splice-switching oligonucleotides (SSOs) capable of altering the splicing of a pre-mRNA encoding a variant of the SLC25A13 gene. The invention also relates to the use of SSOs as therapeutic candidates for treating citrin deficiency. In an aspect of the invention, there is provided a method of exon-skipping comprising providing a splice-switching oligonucleotide (SSO) that binds to a site within a target region present on a pre-mRNA transcript of the SLC25A13 gene, wherein the binding of the SSO induces the exclusion of SLC25A13-PE5 from a mature mRNA transcript of the SLC25A13 gene. In another aspect, there is provided a splice-switching oligonucleotide (SSO) that binds to a site within a target region present on a pre-mRNA transcript of the SLC25A13 gene, the target region having at least 95% sequence identity to SEQ ID NO: 28, and wherein binding of the SSO induces the exclusion of SLC25A13-PE5 from a mature mRNA transcript of the SLC25A13 gene.