74 results about "Insertion site" patented technology

Compositions and methods comprising a deaminase for targeted editing of nucleic acids are provided. Also provided are compositions and methods for localizing a heterologous polypeptide to a target DNA molecule, and compositions and methods for targeted editing of nucleic acids. Fusion proteins comprising an RNA-guided nuclease (RGN) and at least one heterologous polypeptide inserted therein are provided, as well as fusion proteins comprising a DNA binding polypeptide and a deaminase. The heterologous polypeptide may be a pilot editing polypeptide or a base editing polypeptide. Compositions also include nucleic acid molecules encoding a deaminase or fusion protein. Vectors and host cells comprising the nucleic acid molecules encoding the deaminase or fusion protein are also provided.

The application discloses a PCR primer pair and a molecular marker probe for distinguishing between barnyard grass and rice, a detection kit and application thereof. After align analysis on chloroplast genome sequences of the barnyard grass and the rice, it is found that a difference site exists in a 91 kb region, sequences of the upstream and downstream of the difference site are taken as reference sequences to select homologous sequences to design primers for PCR amplification, and the amplification result proves that the difference site fragment obtained by chloroplast genome sequence analysis exists and can be amplified and identified subsequently; the PCR amplification product is sequenced, and the sequencing result is compared to find that a 12 bp insertion site exists in the site and can separate the barnyard grass from the rice, a colloidal gold probe is designed according to the insertion site, and a RPA detection kit and a detection method are established, and the RPA detection kit is used to detect the barnyard grass and the rice, and the barnyard grass and the rice can be accurately distinguished.

Compositions and methods related to transgenic glyphosate tolerant Brassica plants are provided. Specifically, the present invention provides Brassica plants having a DP-073496-4 event which imparts tolerance to glyphosate. The Brassica plant harboring the DP-073496-4 event at the recited chromosomal location comprises genomic / transgene junctions within SEQ ID NO: 2 or with genomic / transgene junctions as set forth in SEQ ID NO: 12 and / or 13. The characterization of the genomic insertion site of the event provides for an enhanced breeding efficiency and enables the use of molecular markers to track the transgene insert in the breeding populations and progeny thereof. Various methods and compositions for the identification, detection, and use of the event are provided.

This invention relates to the field of bioengineering technology, specifically to a recombinant adenovirus displaying a large antigen fragment on its capsid surface, its construction method, and its applications. The HVR1 and HVR5 regions of the hexon hypervariable region of this virus are replaced by gene fragments containing exogenous genes; each exogenous gene fragment has a flexible linker attached to both ends, and the gene sequence of the flexible linker is shown in SEQ ID No. 1. By precisely locating the naturally variable HVR1 and HVR5 loop regions exposed on the surface of the hexon protein as insertion sites, and supplementing them with flexible linkers, the core problem of exogenous sequence insertion disrupting the stability of the hexon structure is successfully solved. This allows for stable packaging to produce viral particles with high titers and infectivity, enabling the development of a new generation of highly efficient and safe capsid-displaying adenovirus vaccines.

The invention belongs to the field of molecular biology and genetic engineering, and discloses a chemiluminescent reporter plasmid pBBR402 and application thereof in detection of the transcription level of a target gene of acetobacter pasteurianus. The plasmid comprises a replication element (oriV and rep) from a pBBR1MCS-2 plasmid, a kanamycin resistance gene from a pMS402 plasmid, a promoter insertion site (BamHI / AvaI / XhoI), a luxCDABE reporter gene cluster and a T1 transcription terminator, and the kanamycin resistance gene, the promoter insertion site (BamHI / AvaI / XhoI), the luxCDABE reporter gene cluster and the T1 transcription terminator are obtained from the pBBR1MCS-2 plasmid. The plasmid can be inserted into a to-be-detected gene promoter of acetobacter pasteurianus to drive luxCDABE expression. During application, the recombinant plasmid is transferred into acetobacter pasteurianus, the OD value and the LUM value of the living bacteria liquid are directly detected, and the transcription level is quantified by the LUM / OD value. The method is easy to operate, high in sensitivity and suitable for gene regulation and control research of acetobacter pasteurianus.

Compositions and methods are provided that include a deaminase for targeted editing of nucleic acids. Also provided are compositions and methods for localizing a heterologous polypeptide to a target DNA molecule and for targeted editing of nucleic acids. Fusion proteins are provided that include an RNA-guided nuclease (RGN) and at least one heterologous polypeptide inserted therein, and fusion proteins that include a DNA-binding polypeptide and a deaminase. The heterologous polypeptide can be a prime editing polypeptide or a base-editing polypeptide. The compositions also include a nucleic acid molecule encoding the deaminase or the fusion protein. Vectors and host cells are also provided that include a nucleic acid molecule encoding the deaminase or the fusion protein.

The invention discloses a lentivirus integration site standard substance and a preparation method thereof, the standard substance is gDNA with clear insertion site and integration frequency, and the standard substance comprises a monoclonal single integration site standard substance, a monoclonal multi-integration site standard substance and a multi-cloning multi-integration site standard substance. The standard substance prepared by the method disclosed by the invention can effectively overcome the defects that a universal detection standard substance is lacked in the market, the accuracy and the sensitivity of the method cannot be verified, and data among laboratories cannot be compared with each other.

A modified vector of adenovirus-associated virus serotype 8 (AAV-8) for gene targeting and expression is provided, wherein the modified vector includes serotype coat amino acid sequence, insertion site and insertion amino acid sequence. A 10-amino acid sequence set forth in SEQ ID NO.3 is inserted between amino acids at positions 590 and 591 that are set forth in SEQ ID NO.2 of the AAV-8 serotype coat protein: LARGDSTKSA, wherein amino acids at positions 1, 2 and 10 are protective amino acids, and amino acids at positions 3 to 9 are screened amino acid sequences. In addition, the present invention also discloses construction method and application of the modified vector are also provided.

This disclosure relates to bicistronic polypeptide constructs for use in allogeneic gene therapy, such as CAR-T cell therapy. A bicistronic construct comprises a first polynucleotide encoding a therapeutic molecule (e.g., CAR-T or antibody) and a second polynucleotide encoding an immune surveillance masking molecule (ISMM). The ISMM includes a non-functional version of a protein knockout, such as a human leukocyte antigen E genetically fused to a fragment, by insertion of a bicistronic construct, such as beta-2 microglobulin or B2M. Also provided are vectors comprising bicistronic constructs, cells (e.g., CAR-T cells), and methods of use. Kits and manufacturing articles are also provided. This disclosure also provides four novel insertion sites that may be used to insert expression constructs into the B2M gene.

Modified E1a regulatory sequences are provided, wherein at least one Pea3 binding site, or a functional portion thereof, is deleted. Also provided are modified E1a sequences that selectively express particular isoforms. Also provided is an E1b-19K clone insertion site. These modified sequences can be used individually, or in combination with one another, to provide tumor-selective expression of proteins.

The invention relates to the technical field of genetic engineering, in particular to identification of a new insertion site of a self-assembled protein cage nanoparticle antigen epitope and application of the new insertion site in vaccine preparation. It is found that a foot-and-mouth disease virus antigen epitope is inserted between 62nd-63rd amino acids of an Encapsulin protein fragment after amino acid sequence modification, and / or the 62nd-63rd amino acids are replaced with the foot-and-mouth disease virus antigen epitope, efficient and soluble expression of a target antigen in escherichia coli can be achieved, and protein cage nano antigen particles are successfully self-assembled; the protein nano antigen particle can induce a widely neutralized anti-foot-and-mouth disease virus antibody, improves the immune efficacy, and has the potential of becoming a foot-and-mouth disease vaccine.

The present disclosure relates to bicistronic polypeptide constructs for use in allogeneic gene therapy, such as CAR-T cell therapy. The bicistronic constructs comprise a first polynucleotide encoding a therapeutic molecule (e.g., CAR-T or an antibody) and a second polynucleotide encoding an immune surveillance masking molecule (ISMM). The ISMs comprise a human leukocyte antigen-E genetically fused to a non-functional form, such as a fragment, of a protein, such as beta-2 microglobulin or B2M, which is knocked out by insertion of the bicistronic construct. Also provided are vectors, cells (e.g., CAR-T cells) comprising the bicistronic constructs and methods of use. Kits and articles of manufacture are also provided. The present disclosure also provides four new insertion sites that can be used to insert an expression construct into the B2M gene.

The application provides a construction and application of a recombinant pseudorabies virus strain stably and efficiently expressing green fluorescent protein, in the research, a green fluorescent protein gene (EGFP) is inserted into different positions of a double gene deletion vaccine strain (JS-2012-△gE / gI) of a pseudorabies virus mutant strain by using a homologous recombination method. Finally, it is proved that the recombinant virus JS-2012-△gE / gI-VP1 / 2-EGFP has good genetic stability, and the EGFP gene can also be stably and efficiently expressed, and the growth rate on cells is also comparable to that of the parent virus, so that the VP1 / 2 gene is the best exogenous gene insertion site. Subsequently, the classical swine fever virus E2 gene, the parvovirus VP2 gene, the cap gene of the circovirus type 2 and the VP1 gene of the foot-and-mouth disease virus can be replaced or inserted into the EGFP expression frame in a single or combined manner to construct different types of multi-vaccines, and the multi-vaccines have extremely high research value and platform significance.

The application discloses a rapid detection method of integration hot spots based on exogenous genetic material insertion sites, uniquely encodes chromosome-site information of all ISs to obtain a digital Y representing the chromosome and the integration position; sorts Ys corresponding to all ISs; for each IS in the insertion site data set, firstly, records the IS as a CIS node C; records other nodes Vi in the insertion site data set; if the distance between C and Vi is less than a threshold TH, records Vi added to the current CIS to become a node of the CIS; simultaneously, connects the edge of C and Vi; then, continues to return to step 2, continues the next IS, and sequentially obtains the CIS network; based on the connection between all ISs, the CIS network is searched to search for a CIS meeting the condition; the application optimizes the connection searching mode and the CIS searching range, and in the detection of several integration sites, the average analysis time of the software is greatly reduced, and the clinical practicability of the CIS detection algorithm is increased.

This invention belongs to the field of biotechnology, specifically relating to a *Pseudomonas putida* genome insertion site library and its construction method. This invention, for the first time, systematically screened and obtained 17 specific genome insertion sites suitable for *Pseudomonas putida*. The constructed insertion site library covers different expression intensities, filling the gap in existing technologies lacking efficient integration sites for *Pseudomonas putida*, and providing diverse options for personalized expression of target genes. The screened insertion sites are all located in the regions of opposing transcription genes, avoiding interference with the host's basal metabolic network. The recombinant strain exhibits stable growth performance without significant growth inhibition, making it suitable for large-scale industrial applications. The integration method, based on homologous recombination, achieves stable integration of the target gene without plasmid maintenance, avoiding expression instability caused by plasmid loss and copy number fluctuations. It also reduces the continuous use of antibiotics, lowering industrialization costs and environmental risks, and the construction method is simple and efficient.

The present disclosure relates to a method of detecting an integration site of a vector in a genome. According to the method of the present disclosure, it is possible to simply and quickly analyze a quantitative integration site of a viral vector with respect to a plurality of DNA motifs (sites) in a genome. Therefore, it can be useful for monitoring the safety and effectiveness of a gene therapy agent.

The invention discloses an eurytopic chlamydomonas reinhardtii expression vector based on a PSAD promoter and a 3 * Flag tag as well as a construction method and application of the eurytopic chlamydomonas reinhardtii expression vector. The nucleotide sequence of the eurytopic chlamydomonas reinhardtii expression vector is shown as SEQ ID No.1; the vector comprises a PSAD promoter, a target gene insertion site EcoR V, a 3 * Flag tag at the 3'terminal, an rbcS2 terminator, a TUB2 promoter, a hygromycin B screening gene and an rbcS2 terminator. The construction method comprises the following steps: constructing a skeleton of a pHyg-PSAD-3Flag vector; constructing a sequence fragment fused with a 3 * Flag tag; and connecting the sequence fragment fused with the 3 * Flag tag with a plasmid skeleton fragment through T4 DNA (deoxyribonucleic acid) enzyme so as to construct the pHyg-PSAD-3Flag vector. According to the invention, the expression plasmid of the chlamydomonas pHyg-PSAD-3Flag can be used for efficiently screening algae strains capable of stably expressing target protein.

Provided are a regulatory element capable of regulating gene expression, and a use. The sequence of the regulatory element is any one or more of sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. By means of innovative bidirectional symmetrical design, the regulatory element can function in two directions at the same time. The regulatory element can be applied to lentiviral vectors, can prevent enhancers / promoters carried by the vectors from affecting downstream genes and prevent uncontrolled and unintended expression caused by enhancers / promoters of host genes at insertion sites, and thus can improve the safety of vector applications.

Provided are a highly efficient base editing system, a preparation method therefor and the use thereof. Specifically, provided is a fusion protein, comprising a nuclease domain and an editing enzyme domain, wherein the editing enzyme domain is inserted into an insertion site in the nuclease domain. The nuclease domain in the fusion protein has the function of a nuclease, the editing enzyme domain in the fusion protein has the function of an editing enzyme, and the nuclease domain is derived from a Cas protein or a Cas protein mutant. On the basis of an activation-induced cytosine deaminase (AID), a new AID mutant having a shorter sequence and higher base editing efficiency is obtained by means of screening, and then the highly active AID mutant is randomly inserted into nSaCas9 (D10A) KKH, such that a new cytosine base editor EB-TAM having high base editing efficiency and an expanded and narrowed editing scope is successfully obtained by means of screening.

A novel transgenic corn elite event designated MZIR260 is disclosed. The invention relates to DNA sequences of the recombinant constructs inserted into the corn genome and of genomic sequences flanking the insertion site that resulted in elite event MZIR260. The invention further relates to assays for detecting the presence of the DNA sequences of corn elite event MZIR260, to corn plants and corn seeds comprising the genotype thereof, and to methods for producing a corn plant by crossing a corn plant comprising the elite event MZIR260 genotype with itself or another corn variety.

The present invention relates to a novel retroviral vector comprising a 5'long terminal repeat (LTR), a primer binding site, a heterologous intron, a transgene insertion site and a 3 'LTR wherein the heterologous intron is located immediately downstream of the 5' LTR. This vector has high titer productivity and is particularly suitable for gene therapy.

This application provides a protein stability detection probe based on ascorbate peroxidase 2 and its application. The detection probe includes ascorbate peroxidase 2 displayed on the cell surface and an anchoring protein that anchors ascorbate peroxidase 2 to the cell. Ascorbate peroxidase 2 has an insertion site for the target protein, into which the target protein inserts. Ascorbate peroxidase 2 is divided into N-terminal and C-terminal ascorbate peroxidase 2, and the target protein is linked to both ends of the C-terminal ascorbate peroxidase 2 via flexible linkers. The intensity of the fluorescence signal generated by the cascade reaction catalyzed by the probe in this application has a good linear relationship with the stability of the inserted test protein mutant, enabling high-throughput identification of protein mutant stability and showing promising application prospects.

The invention relates to the technical field of genetic engineering, in particular to Encapsulin fusion protein for expressing foot and mouth disease virus epitopes and application of the Encapsulin fusion protein in preparation of subunit vaccines. The invention discovers that different foot-and-mouth disease virus antigen epitopes are inserted among the 62nd to 63rd positions, the 124 to 125th positions, the 138 to 139th positions and the 239th to 240th positions of amino acids of an Encapsulin protein fragment subjected to amino acid sequence modification, and / or the 62nd to 63rd positions, the 124 to 125th positions, the 138 to 139th positions and the 239th to 240th positions of amino acids are replaced by different foot-and-mouth disease virus antigen epitopes; efficient and soluble expression of a target antigen in escherichia coli can be realized, and protein cage nano antigen particles are successfully self-assembled; the protein nano antigen particle can induce a widely neutralized foot-and-mouth disease virus antibody, improves the immune efficacy, and has the potential of becoming a broad-spectrum foot-and-mouth disease vaccine.

A novel transgenic corn elite event designated MZIR098 is disclosed. The invention relates to DNA sequences of the recombinant constructs inserted into the corn genome and of genomic sequences flanking the insertion site that resulted in elite event MZIR098. The invention further relates to assays for detecting the presence of the DNA sequences of corn elite event MZIR098, to corn plants and corn seeds comprising the genotype thereof, and to methods for producing a corn plant by crossing a corn plant comprising the elite event MZIR098 genotype with itself or another corn variety.

Polypeptides are disclosed having an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence selected from SEQ ID NO: 1-28, not including any amino acid insertions at identified insertion sites, wherein the polypeptide binds to the F 1 domain of insulin receptor; or SEQ ID NO: 29-31, wherein the polypeptide binds to the LI domain of insulin receptor; fusion proteins thereof, and. methods for their use.

The present disclosure provides a transposon molecular marker for identifying Oryza sativa subsp. indica and its application. The transposon molecular marker is first provided as a specific transposon insertion marker for Oryza sativa subsp. indica, and the insertion site is located between the 39,031,819th and 39,031,820th nucleotides of chromosome 1 of the Oryza sativa japonica reference genome version IRGSP-1.0. The variety of Oryza sativa subsp. indica contains a 259 bp transposon insertion at the insertion site, while the variety of other Oryza sativa subspecies does not contain the transposon insertion at the insertion site. The application of the above transposon molecular marker is further provided. The transposon molecular marker for identifying Oryza sativa subsp. indica of the present disclosure has the advantages of strong specificity, high accuracy, simple operation, and being not affected by the environment, and can be applied to Oryza sativa subsp. indica germplasm identification, molecular marker assisted breeding, and rapid seed purity detection, and has important practical value.

The invention relates to the technical field of synthetic biology, and discloses an antibiotic-label-free genome traceless editing method in blue-green algae, which comprises the following steps: S1, constructing a chassis cell: knocking out a nitrate reductase gene of the blue-green algae to obtain a mutant strain which can only grow by taking nitrite as a sole nitrogen source; s2, constructing a plasmid platform: constructing at least one recombinant plasmid containing a nitrate reductase gene, a homologous arm and a target gene insertion site; s3, target gene insertion: cloning an exogenous target gene to the target gene insertion site of the recombinant plasmid in the step S2 to obtain a recombinant expression plasmid containing the target gene; s4, transformation and screening: introducing the recombinant expression plasmid in the step S3 into the chassis cell in the step S1, and screening a positive transformant through a nitrate-containing culture medium to realize traceless insertion of an exogenous gene; and S5, repeating the steps S2, S3 and S4, and realizing continuous traceless insertion of a plurality of exogenous genes by alternately using a culture medium containing nitrate or nitrite.

Provided herein are methods of high-throughput identification to identify sites for transgene integration in the genome of biological samples. The method integrates a self-recording nucleic-acid cassette across the genome in a massively parallel manner, and the recording is activated by introducing Cas9 protein into the system. Quality of the insertion site is determined by analyzing the number of mutations by recording process in a polyclonal expansion group of cells in a biological sample.

This invention provides a method for rapidly identifying exogenous inserted sequences based on whole-genome sequencing data, comprising the following steps: sequencing data quality control, preliminary data alignment, screening of preliminary alignment results, evaluation of secondary alignment results, homology detection, preliminary insertion region detection, precise insertion site identification, assembly of inserted and flanking sequences, acquisition of genes affected by the insertion site, and primer design for the insertion site. Compared with traditional experimental detection techniques, this invention is less time-consuming, reproducible, and presents more comprehensive transgenic insertion characteristics; compared with other existing whole-genome resequencing methods, it is more accurate and intuitive, the results are easier to understand, and it is more conducive to the safety risk assessment of transgenic plants and animals, thus contributing to the development and application of transgenic or gene editing technologies.

The compositions and methods disclosed relate to DNA compositions, plant cells, seeds, plant parts that relate to maize maintainer plants. Also provided are assays for detecting the presence of the maize DP-056113-9 event based on the DNA sequence of the recombinant DNA construct inserted into the maize genome and the DNA sequences flanking the insertion site. Kits and conditions useful in conducting the assays are provided.