127 results about "Insertion site" patented technology

The invention belongs to the technical field of gene sequencing, and particularly relates to a method for efficiently identifying an exogenous DNA insertion site in a genome by using a high-throughput sequencing technology. The method comprises the following steps: (a) extracting a sample, building a database and sequencing; (b) data quality control; (c) comparing data; (d) filtering comparison results; (e) preliminary screening; and (f) screening and identifying the positions of the exogenous insertion elements. According to the method, the high-throughput sequencing data and the genome and exogenous fragment sequences are compared and analyzed to obtain the candidate positions of the insertion sites, so that the test efficiency can be remarkably improved, manual judgment and screening work can be effectively replaced, and the result accuracy can be improved.

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 invention discloses a construction method and application of an iPSC-derived mesenchymal stem cell strain for overexpressing a VEGFA gene based on a CRISPR / Cas9 technology, and relates to the technical field of biological cells, the construction method of the iPSC-derived mesenchymal stem cell strain for overexpressing the VEGFA gene based on the CRISPR / Cas9 technology comprises the following steps: S1, designing sgRNA and ssDNA for an insertion site AAVS1, and detecting the cutting efficiency of the sgRNA; s2, introducing a CRISPR (clustered regularly interspaced short palindromic repeats) system into the cells in an electroporation manner to obtain an iPSC cell strain into which an exogenous gene VEGFA is inserted at a fixed point; s3, carrying out directional induced differentiation on the iPSC cell strain to the iMSC to obtain an iMSC cell strain with overexpression of VEGFA (Vascular Endothelial Growth Factor A) protein; the iMSC of the overexpressed VEGFA protein can well stimulate vascular endothelial growth and promote angiogenesis, and more possibilities are provided for repair treatment of ischemia-related diseases.

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.

The present invention discloses the development and application of a genomic editing tool for phytopathogenic bacteria. The present invention provides a DNA molecule I, which has element A, element B, and element C; element A is an expression cassette for expressing the ligD gene and the Ku gene; element B is an expression cassette for expressing the Cas12a gene; the element C has an insertion site and a DNA segment for encoding the crRNA backbone; the insertion site is for inserting a DNA segment for encoding the specific binding region of the crRNA; the specific binding region of the crRNA specifically binds to the target sequence. The present invention constructs a set of genomic editing tools suitable for phytopathogenic bacteria by fusing the ligD / Ku protein with the CRISPR / FnCas12a system. This method adds a new powerful tool to the genetic analysis toolbox of phytopathogenic bacteria and has great value for studying the molecular interaction mechanism between host plants and pathogenic bacteria.

The invention discloses a method for detecting an insertion site of agrobacterium T-DNA on a plant genome, which comprises the following steps: carrying out sequence merging on a T-DNA genome and a plant genome to obtain a merged genome G; performing sequencing read sequence alignment on the combined genome G to obtain an SAM file with an alignment result; performing classification analysis on the SAM file with the comparison result; sequentially carrying out blacklist filtering, merging and repeated area marking analysis on a classification analysis result; and finally outputting the number of the final integration sites and the number of the real integration events. According to the method disclosed by the invention, the sensitivity of T-DNA integration site detection is increased and the false positive rate of detection is reduced by taking measures of considering the special condition of double-end sequencing of an insertion site part, establishing a blacklist, carrying out secondary matching optimization on a specific repetitive sequence of a plant genome, carrying out multi-sequence similarity comparison on a result site and the like.

The invention belongs to the technical field of biomolecular diagnosis, particularly relates to an identification method of a transposon insertion site, and aims to solve the problem that an existing method cannot provide accurate and comprehensive information of the transposon insertion site. The method comprises the following steps: transferring an MTD artificial transposon with specific DNA Barcode, gRNA and CRISPR / Cas system gene editing tool enzyme to a target cell, carrying out gene editing on the target cell, the MTD artificial transposon comprising a sequence represented by SEQ ID NO: 1; and identifying the position information of the specific DNA Barcode through PCR (Polymerase Chain Reaction) amplification and sequencing or probe hybridization so as to obtain the insertion position of the transposon. The specific DNA Barcode is marked on the MTD transposon, and the accurate insertion position of the MTD transposon can be obtained by detecting the DNA Barcode.

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.

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.

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 invention belongs to the field of genetic engineering, and particularly relates to a genetically engineered bacterium for high yield of lipase and a preparation method and application of the genetically engineered bacterium. The starting strain of the genetically engineered bacterium is pichia pastoris, and lipase genes are inserted into the genome of the pichia pastoris; and the insertion site of the lipase gene is between the PASchr31156 gene and the PASchr30154 gene on the No.3 chromosome of the pichia pastoris or between the PASchr40465 gene and the PASchr40467 gene on the No.4 chromosome of the pichia pastoris. The lipase ROL is produced by using a pichia pastoris eukaryotic expression system, and the secretory expression level of the lipase in pichia pastoris is improved by adjusting the integration site of the lipase ROL gene in the pichia pastoris genome, optimizing the copy number and adding the molecular chaperone, so that a better effect is achieved; the yield of the lipase high-producing strain prepared by the invention reaches 8.42 mg / mL, and the lipase high-producing strain has a relatively good enzyme activity level, and overcomes the defect that the expression quantity of related lipase expressed by existing pichia pastoris gene engineering bacteria is relatively low.

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.

A use of an SRE-PTPRZ1 inhibitor or a PTPRZ1 inhibitor in the preparation of a drug for treating squamous cell carcinoma. The SRE-PTPRZ1 inhibitor is an inhibitor for SRE-PTPRZ1 structural variation, the SRE-PTPRZ1 structural variation being somatic simple repeat expansion at insertion site GRCh38 chr7: 121603263-121603506, the repeating pattern of an insertion region and a somatic cell simple repeat expansion sequence are (CTTT)n, wherein 10 ≤ n < 500; the PTPRZ1 inhibitor is one of the followings compounds, a pharmaceutically acceptable salt thereof, one or two derivatives among derivatives thereof, or a mixture of the above: NAZ2329, SCB4380, MY-33-3, or MY-10; and the HGNC number of the PTPRZ1 gene is 9685.

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 invention provides a method for enhancing gene knock-in efficiency mediated by a CRISPR gene editing system in a cell. The method comprises the step of enabling the cell to be in contact with a DNA-dependent protein kinase (DNA-PK) inhibitor and a cell division cycle protein 7 (Cdc7) inhibitor in the gene knock-in operation process of the cell. The invention also provides a kit for enhancing the gene knock-in efficiency mediated by the CRISPR gene editing system in cells. According to the method and the kit provided by the invention, the DNA-PK inhibitor and the Cdc7 inhibitor are combined for use, so that the gene knock-in efficiency can be remarkably improved under the conditions of different cell lines, different insertion sites and different donor template lengths.

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 the field of molecular biology, and in particular to a primer set for detecting rice MITE transposon insertion sites and its application. The primer set includes 16 specific primers, and the sequence is shown in SEQ ID NO: 11-26; by using any single primer or a mixed combination of any primers in the primer set, through library construction PCR, and then NGS second-generation deep sequencing, about 29,000 MITE sites can be enriched from each rice material, and the detection rate exceeds 80%; the present invention provides a powerful tool for rice MITE site detection, and has important technical guiding significance for the fields of rapid rice molecular breeding, germplasm identification, etc.

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.

The present disclosure relates generally to compositions, methods and systems for characterizing a biological particle, cell or cell nucleus, modified to include an exogenous insert, e.g., exogenous nucleic acids. The characterization may identify the insertion site of the exogenous nucleic acids in the biological particle's endogenous, e.g., genomic, nucleic acid sequences. Such characterization may be useful in the development of safer, more effective, modified cell biotherapeutics. e.g., cells modified to express chimeric antigen receptors.