49 results about "Exogenous DNA" patented technology

The invention discloses a TaIMPA1 gene and application of the TaIMPA1 gene in improvement of plant genetic transformation efficiency. The nucleotide sequence of the gene is shown as SEQ ID No: 1 in a sequence table. According to the invention, the key gene TaIMPA1 is cloned from a plant body to delivery for the first time, and the genetic transformation performance is enhanced by remarkably improving the delivery efficiency of exogenous DNA (Deoxyribose Nucleic Acid). The discovery fills up the research blank of plant endogenous delivery regulatory factors, and an original solution is provided for overcoming the core problem of genotype dependence.

The invention belongs to the technical field of agricultural biology, and particularly relates to a plant constitutive promoter OsSULTR2. 2pro and an application thereof. The promoter is characterized in that the promoter is OsSULTR2; the nucleotide sequence of the 2pro is as shown in SEQ ID NO. 1. The promoter provided by the invention is OsSULTR2; 2pro is a rice endogenous constitutive promoter, can drive a target gene to realize efficient and stable expression in plant callus, roots, stems, leaves, young ears, seeds and other tissues in a vegetative growth period, and can replace an existing non-plant-source promoter. The method has important application value in the field of plant genetic engineering, and can effectively reduce potential safety risks of transgenic plants caused by introduction of exogenous DNA.

The invention provides a method for cultivating anti-aging glutinous highland barley, highland barley starch as well as a preparation method and application of the highland barley starch, and particularly relates to a method for improving the starch quality character of an existing high-quality glutinous highland barley variety, in particular to a method for cultivating anti-aging glutinous highland barley by utilizing a gene editing technology without exogenous DNA (Deoxyribose Nucleic Acid) integration. A method for fixed-point knockout of an SSIIa gene to cultivate an anti-aging glutinous highland barley new strain under the background of a "Zhengnongglutinous 232" variety comprises the following steps: providing an acceptor material derived from a highland barley variety "Zhengnongglutinous 232" with a glutinous character, and deleting a Wx gene function of the "Zhengnongglutinous 232"; the method comprises the following steps: assembling guide RNA (Ribonucleic Acid) designed aiming at an SSIIa gene and Cas9 protein in vitro to form a ribonucleoprotein RNP complex; introducing the RNP complex into cells of an acceptor material so as to enable Cas9 protein to generate double-chain breakage at a target site of the SSIIa gene; carrying out culture and plant regeneration on the introduced cells to obtain a T0-generation plant of which the SSIIa gene is edited; and screening plants with the function loss of the SSIIa gene, wherein the plants keep the glutinous character of adult agricultural glutinous 232.

The invention relates to a method for splitting, assembling and site-specific integration of large-fragment exogenous DNA (deoxyribonucleic acid) in Trichoderma reesei. Comprising the following steps: (1) constructing an arg:: Ptcu-Cas9-TtrpC-hph strain containing a Cas9 protein; (2) constructing a pUC19 <-> integration site: a pyr4 plasmid; (3) obtaining a plurality of target gene segments containing integration site homologous arms and having overlapping regions at the tail ends of adjacent DNA segments; (4) obtaining a gRNA expression cassette of a targeted genome integration site; (5) arg conversion: the arg is converted into a Ptcu-Cas9-TtrpC-hph strain; and (6) screening and verifying. According to the method, the CRISPR / Cas9 system is combined with the TAR in the trichoderma reesei for the first time, the exogenous large-fragment DNA is split into a plurality of small fragments, then the plurality of small-fragment exogenous DNA are assembled in the trichoderma reesei and are subjected to fixed-point integration, one-step editing of the large-fragment exogenous DNA is realized, and compared with a traditional genetics method, the gene editing efficiency in the trichoderma reesei is greatly improved.

This invention discloses a gene editing method and application for *Trichoderma reesei* based on a ribonucleoprotein complex. It aims to overcome the problems of low editing efficiency, easy generation of heterozygotes, and risk of exogenous DNA integration in *Trichoderma reesei* using plasmid-based CRISPR / Cas9 systems. This invention combines a dual-site cleavage strategy guided by double sgRNAs with direct RNP delivery. The method involves: designing two guide RNAs spaced 50-700 bp apart for the target gene, pre-assembling them with Cas9 protein in vitro to form an active RNP complex; delivering this complex along with exogenous donor DNA to *Trichoderma reesei* protoplasts. The exogenous donor DNA contains a left homologous arm, an editing sequence, and a right homologous arm, wherein the left and right homologous arms are homologous to the genomic sequences outside the dual cleavage sites on the target gene determined by the double sgRNAs, respectively; upon entering the cell, the RNP complex introduces dual-site cleavage at the target site, achieving precise editing under the guidance of the exogenous donor DNA.

Provided is a means for constructing a modified genomic DNA which has a nucleotide sequence different from an existing genomic DNA sequence, contains a modified gene capable of expressing a polypeptide, a functional RNA, or both, and does not contain exogenous DNA.　This method comprises: performing, at least twice, (a) a step for specifically cleaving genomic DNA of a cell into at least two target sequences by means of a sequence-specific nuclease, and linking the resultants through a DNA double-stranded cleavage repair by the cell; and deleting at least two regions from an existing gene and non-coding regions adjacent thereto to thereby produce a modified gene.

The invention discloses a filamentous fungus genetic transformation system taking an endogenous KatG1 gene as a selection marker, a method and application. The method comprises the following steps: carrying out linear DNA fragment construction on an endogenous Pgpd promoter, an endogenous KatG1 gene, a target promoter and a target gene of filamentous fungi, then transforming the constructed linear DNA fragment into filamentous fungi cells, and finally screening through a menadione-containing culture medium to obtain the directionally improved filamentous fungi strain containing the target gene. According to the invention, the endogenous KatG1 gene of the fungus is used as a selection marker gene and menadione is used as screening resistance to construct a novel selection marker based on an endogenous gene and a genetic transformation system without exogenous DNA (Deoxyribose Nucleic Acid) such as a carrier and the like, so that the method has the advantages of universality, high efficiency and safety; an environment-friendly solution is provided for directional genetic improvement of filamentous fungi, and the method has important commercial application potential in directional breeding of fungus strains in the fields of agriculture, industry and the like.

The invention belongs to the technical field of animal genome editing, and particularly relates to a method for realizing fixed-point integration of full-length exogenous DNA fragments by using long single-stranded DNA. The technical problem to be solved by the invention is to provide a method for efficiently realizing site-specific integration of an exogenous full-length gene in a mammal genome. According to the technical scheme, the method for realizing fixed-point integration of the full-length exogenous DNA fragment by using the long single-stranded DNA comprises the following steps: converting a mixture of sgRNA, Cas9mRNA and / or Cas9 protein of a long single-stranded exogenous gene and guiding Cas9 to cut an integration site into recipient cells; the exogenous gene long single strand comprises a 5'homologous arm and a 3 'homologous arm which are arranged on the upstream and downstream of an sgRNA guide cleavage site, a full-length exogenous DNA fragment sequence arranged between the 5' homologous arm and the 3 'homologous arm, and related matching original parts required by expression of the full-length exogenous DNA fragment sequence.

The invention discloses a construction method and application of a kluyveromyces marxianus mutant, and belongs to the field of synthetic biology and molecular biology. According to the method, single-stranded DNA binding protein and cytosine deaminase pmCDA1 are fused, a random base editing system is constructed, and whole-genome efficient C-T / G-A mutation is realized in kluyveromyces marxianus by taking MCM2-7 as a verification target. The system does not need a specific target or an exogenous DNA template, the mutation efficiency is high, the distribution is wide, the mutation rate is improved by 303.1 times compared with that of a control group through optimal combination, and the application potential of kluyveromyces marxianus in industrial production is expanded. The method is combined with an efficient screening system, mutant strains with the intracellular protein expression quantity remarkably improved are screened out, the constructed mutation system is not only suitable for kluyveromyces marxianus cell factory transformation, but also can be popularized to genome evolution of other yeasts or eukaryotic hosts, and a new strategy is provided for industrial microorganism breeding and protein engineering.

This invention belongs to the field of agricultural biotechnology, specifically relating to a plant constitutive promoter OsSULTR2;2pro and its applications. The nucleotide sequence of the promoter OsSULTR2;2pro is shown in SEQ ID NO.1. The promoter OsSULTR2;2pro of this invention is an endogenous constitutive promoter for rice, capable of driving efficient and stable expression of target genes in plant callus tissue, roots, stems, leaves during vegetative growth, as well as young panicles and seeds, and can replace existing non-plant-derived promoters. It has significant application value in the field of plant genetic engineering and can effectively reduce the potential safety risks of transgenic plants caused by the introduction of exogenous DNA.

The invention belongs to the technical field of agricultural biology, and particularly relates to a rice endogenous space-time specific promoter OsSULTR1; the invention discloses 3pro and application thereof. The promoter OsSULTR1 is characterized in that the promoter OsSULTR1 The nucleotide sequence of 3pro is as shown in SEQ ID NO. 1. The invention provides an OsSULTR1; 3pro is a space-time specific expression regulation element from rice, can efficiently and stably drive a target gene to be expressed in a plant callus, and can realize targeted efficient and stable expression in root and stem tissues in a plant vegetative growth period. The promoter can effectively replace a non-plant-source promoter commonly used in existing plant genetic engineering, has an important industrial application value in the field of plant genetic engineering, and not only can improve the targeting and stability of target gene expression in transgenic plants, but also can improve the yield of the transgenic plants. And the potential biological safety risk of the transgenic plant caused after the exogenous DNA is introduced into the plant genome can be obviously reduced.

The invention discloses a method for creating high-anthocyanin tomatoes through a gene knock-in technology. A homologous recombination mediated gene knock-in technology is utilized, the Aft gene of the tomato is knocked into the downstream of a promoter of a fruit ripening specific gene E8 at a fixed point, the E8 promoter is successfully utilized to activate Aft gene expression, and the high-anthocyanin tomato with purple black tomato fruit is obtained; and further performing selfing on the high-anthocyanin tomatoes, and screening out non-transgenic high-anthocyanin tomatoes which are subjected to Aft gene homozygous insertion and do not carry exogenous DNA fragments from selfing filial generations of the high-anthocyanin tomatoes. Therefore, the gene editing method disclosed by the invention can be used for quickly converting the common tomato without accumulation of anthocyanin into a high-nutrition tomato material rich in anthocyanin, and has great breeding application prospect and economic value.

The present invention relates to the field of biotechnology and specifically discloses a method for transforming Rhodosporidium toruloides. The method comprises the following steps: 1) obtaining a single clone of Rhodosporidium toruloides, inoculating it into a culture medium for cultivation, and collecting the cells; 2) adding a transformation solution to the cells in step 1), resuspending the cells, and allowing them to stand; the transformation solution comprises PEG, a mediator, and salmon sperm DNA; 3) adding the DNA to be transformed to the cells in step 2), and incubating the cells; 4) adding a dithiothreitol solution or a dimethyl sulfoxide solution to the cell solution in step 3), followed by heat shock, resuspending the collected cells, and incubating them. The present invention constructs an efficient and reliable method for transforming Rhodosporidium toruloides with exogenous DNA. The transformation method of the present invention for Rhodosporidium toruloides has a simple process and high transformation efficiency, with a maximum transformation efficiency of 1938 transformants / μg DNA.

Methods are provided for determining the genomic location of a genomically integrated exogenous DNA. The methods can use nuclease agents to create an enriched library for long- read sequencing to provide hybrid alignments to both a reference genome and the exogenous DNA sequence, thereby determining the location of the genomically integrated exogenous DNA.

The present application belongs to the field of microbial metabolic engineering and industrial microorganism technology, and particularly relates to a method for stably integrating exogenous DNA into Pseudozyma roseolata, an iterative gene editing method and application thereof. The preservation number of the strain MYL-2 is CCTCC M 20252968, and the strain can efficiently accumulate oil under nitrogen-limited culture conditions, the main fatty acid components in the cells are C16-C18 fatty acids, and the strain has application potential as a microbial oil production strain. The present application provides a method for stably integrating exogenous DNA into Pseudozyma roseolata, and constructs a recombinant expression vector pNFH-loxp-RAD51 and a Cre recombination vector containing a loxP site, and for the first time, precise removal and repeated use of a screening marker mediated by a Cre-LoxP system are realized in the strain, thereby laying a foundation for multi-round gene editing and efficient engineering modification of the strain.

The present application relates to the field of agricultural biotechnology, and particularly relates to a CEPro722 promoter and application thereof.The CEPro722 promoter comprises a nucleotide sequence as shown in SEQ ID NO.1 or a nucleotide sequence as shown in SEQ ID NO.2.The present application is screened from a GRMZM2G069722 gene, and the CEPro722 promoter can drive gene expression in plants such as rice, wheat or corn; and particularly, the promoter can drive high and stable expression in various tissues such as plant callus, leaf in the vegetative growth period, young ear and seed, and can replace a non-plant source promoter.The CEPro722 promoter provided in the present application has important significance in the field of plant genetic engineering, and can effectively reduce potential safety risks of transgenic plants caused by exogenous DNA.

The application discloses a detection method based on dominant clones caused by exogenous DNA insertion mutation, and the method comprises the following steps: 1, preprocessing of virus IS information data by positive and negative strand correction and redundant integration; 2, after the preprocessing is completed, IS caused by non-specific amplification is identified to remove false positive IS detection results caused by PCR non-specific amplification; 3, according to a site clustering algorithm, IS in adjacent positions is combined and clustered; 4, according to the clustering results, a clone plane is analyzed, and dominant clones are described; and the application utilizes a PCR primer similarity ranking algorithm to identify and remove non-specific amplification caused by possible PCR primer similarity, so that the false positive of IS identification is significantly reduced in actual application, and the accuracy is improved.

The present invention is directed to a genome editing kit for targeted integration of an exogenous DNA in a target site of a large gene by non-homologous end joining (NHEJ) or by microhomology-mediated end joining (MMEJ), wherein said kit comprises donor nucleic acids for replacing the 5' and 3' portions of the target gene relative to the target site.

The invention discloses a method for targeted blocking of antibiotic resistance gene horizontal transfer based on replication-defective engineering bacteriophage and application, and belongs to the technical field of environmental microbiology and gene engineering. In order to solve the problems of ARGs release caused by a splitting strategy and biosafety risk of the engineering bacteriophage, the replication-defective engineering bacteriophage obtained by packaging a CRISPR-Cas9 system with a targeted cleavage resistance gene through a packaging system related to a bacteriophage display technology can reduce the risk of drug-resistant gene diffusion in an environment and a medical scene. The method has the main advantages that: 1) dual specificity: the method has the recognition capability of phage on specific host phenotype and the specific recognition of CRISPR-Cas9 on ARGs sequence, and dual precise targeting is realized; 2) the biosafety is high: after the element is delivered, the element cannot be copied in a host, the exogenous DNA is easily degraded by the host, and the residue and unexpected evolution of the engineering bacteriophage in the environment are avoided;

The application belongs to the field of agricultural biotechnology, and particularly relates to a plant constitutive promoter OsSULTR1;2pro and application thereof. The nucleotide sequence of the promoter OsSULTR1;2pro is shown as SEQ ID NO. 1. The promoter OsSULTR1;2pro is an endogenous constitutive promoter of rice, and can drive efficient and stable expression of a target gene in plant callus, roots, stems, leaves and grains in the vegetative growth period, and can replace existing non-plant origin promoters. The application has important application value in the field of plant genetic engineering, and can effectively reduce potential safety risks of transgenic plants caused by introduction of exogenous DNA.

The invention provides a method for genetically modifying a cell by using a lambda Red recombination technology, which comprises the following steps: (a) preparing a cell to be modified, namely culturing the cell with a lambda Red recombination system at a first temperature until OD600 is equal to 0.1-0.2; inducing expression of a lambda Red recombination system in the cells, and culturing the cells at a second temperature until OD600 = 0.4-1.5, the second temperature being at least 4 DEG C higher than the first temperature; cooling and centrifuging the cells to obtain a cell block mass; resuspending the cell mass with a glycerin solution to obtain a to-be-modified cell suspension; (b) preparing a targeting fragment, comprising: linearizing a plasmid comprising an exogenous DNA fragment; amplifying the exogenous DNA fragment by taking the linearized plasmid as a template; and recovering an amplification product as a targeting fragment, wherein the targeting fragment comprises the exogenous DNA fragment and homologous arms positioned on two sides of the exogenous DNA fragment; and (c) introducing the targeting fragment obtained in the step (b) into the to-be-modified cell obtained in the step (a), and continuously culturing the cell at the temperature of not less than 35 DEG C.

The application discloses TaIMPA1 A gene and application thereof in improving plant genetic transformation efficiency. The nucleotide sequence is shown in the sequence table SEQ ID No: 1. The application clones the key gene from a plant body for the first time TaIMPA1 which enhances the genetic transformation performance by significantly improving the efficiency of exogenous DNA delivery. The finding fills the research blank of endogenous delivery regulatory factors in plants and provides an original solution to the core problem of overcoming genotype dependence.

The invention discloses a herbicide-resistant alfalfa transformation event TWMS2-17 and a detection method thereof, an exogenous DNA fragment is inserted into an alfalfa genome, and herbicide-resistant plants TWMS2-17 are obtained through herbicide spraying treatment and single-copy plant screening. Through analysis and verification of a flanking sequence of a T-DNA insertion site, the position of an exogenous gene inserted into a genome is a No.3 chromosome, a 270bp genome sequence is replaced, and after asexual cuttage planting, excellent glyphosate and glufosinate resistance is shown, and normal growth is realized. The DNA molecule of the transformation event and the detection method have remarkable application value on breeding of herbicide-resistant alfalfa.

The invention belongs to the technical field of plant genetic transformation, and discloses a liposome-mediated maize pollen transfection method. The method comprises the following steps: preparing a plasmid DNA solution and a mixed lipid vesicle SMV solution composed of lecithin and cationic lipid DOTMA, and mixing and incubating the plasmid DNA solution and the mixed lipid vesicle SMV solution according to a mass ratio to form an SMV-DNA compound transfection solution; collecting and pretreating fresh corn pollen, and co-incubating with the transfection solution at low temperature; after drying treatment, the pollen is directly used for pollinating female ears of receptor corn, and transgenic offspring seeds are obtained. According to the method, efficient delivery of exogenous DNA is realized by utilizing affinity and electrostatic interaction of liposome and pollen cell membranes, tissue culture is not needed, the method is independent of genotypes, the method is simple and convenient to operate and low in cost, the problem of somatic cell variation is effectively avoided, the transformation efficiency can reach about 1.4%, and an efficient and practical new technical approach is provided for corn genetic improvement.

The invention discloses a method for editing a Transgene-free (Transgene-free) gene. The Transgene-free gene editing method disclosed by the invention comprises the following steps: 1) inserting a spacer sequence of a targeted target gene into a pHSC plasmid to obtain a recombinant vector which can transcribe sgRNA of the targeted target gene; and 2) infecting the biological material with recombinant agrobacterium containing a recombinant vector, and then carrying out enrichment culture on the obtained biological material in a culture medium containing hygromycin for 7-25 days to obtain a biological material in which a target gene is edited and which does not contain exogenous DNA, namely, realizing Transgene-free gene editing of the target gene. According to the method disclosed by the invention, the proportion of the transgene-free editing cells is greatly increased, so that the time and the workload of subsequently screening the transgene-free editing mutant are greatly reduced, and the method has a very good application prospect.

The invention relates to an in vitro method for modifying double stranded DNA (dsDNA) in a eukaryotic cell. The method comprises: a) introducing a base editor system into the cell, said system comprising i) a base editor, comprising an RNA guided DNA nickase linked to a single-stranded DNA nucleobase modifying enzyme, or a nucleic acid encoding said base editor, and ii) at least two guide RNA (sgRNA) molecules, comprising a first sgRNA that hybridizes to a first target sequence in the dsDNA, and a second sgRNA that hybridizes to a second target sequence in an opposing strand of the dsDNA, b) generating at least two single-strand breaks (SSBs), in opposing strands, of the dsDNA, wherein a first base editor complex, comprising the first sgRNA, introduces a nick cleavage of one strand of the dsDNA in the first target sequence, and a second base editor complex, comprising the second sgRNA, introduces a nick cleavage of the opposite strand of the dsDNA in the second target sequence, thereby producing a 5' or 3' overhang, c) inserting an exogenous DNA repair template sequence in proximity to the two single strand breaks (SSBs). The invention further relates to a genetically modified eukaryotic cell obtained by the method according to the present invention. The invention also relates to a kit for use in the in vitro method.

The invention provides a method for identifying and eliminating pollutants during quantification of microbe cfDNA in peripheral blood, which comprises the following steps: S1, screening common bacteria of peripheral blood and esophageal-gastric junction tumor, and extracting bacterial nucleic acid; s2, designing a corresponding primer probe combination according to the specific nucleic acid sequence of the bacteria, and carrying out multiple amplification on the bacterial nucleic acid by utilizing fluorescent quantitative PCR (Polymerase Chain Reaction); s3, carrying out statistical classification on the amplified specific nucleic acid sequence in the step S2 by utilizing R statistical software decontam to obtain a statistical classification program; the method comprises the following steps: adding artificially synthesized exogenous DNA during preparation of a sample of microorganism cfDNA in peripheral blood, and setting an identification sample confusion method; and S4, identifying and eliminating pollutants through a statistical classification program, and quantifying the microbe cfDNA in the peripheral blood. The method disclosed by the invention can be used for accurately, quickly and conveniently detecting the bacterial nucleic acid in the peripheral blood, identifying and eliminating polluted samples at the same time, and avoiding false positive during detection.

This invention relates to a novel engineered restriction-modification system for salt-tolerant Jeju bacteria to improve the efficiency of exogenous DNA transformation, belonging to the fields of bioengineering, metabolic engineering, and synthetic biology. This invention involves in-depth analysis of the whole-genome methylation map of Jeju bacteria to identify its unique methylation motif SAGCTS, and further identifies the corresponding restriction endonuclease Sen. Based on this, the restriction-modification system of Jeju bacteria is edited and modified, thereby improving the efficiency of exogenous DNA transformation.

The invention discloses an application of a small molecule compound PJ34 in improvement of leader editing efficiency, the leader editing efficiency can be enhanced by adding PJ34, the method is simple and convenient to operate, modification of leader editor protein or pegRNA is not needed, and testing of different pegRNA sequences through a large number of pre-experiments is not needed. In addition, PJ34 is easy to synthesize and low in price, and has no obvious toxic effect on cells under the concentration of improving the editing efficiency. Compared with other methods for enhancing the editing efficiency, the method for enhancing the editing efficiency by adding the PJ34 avoids transformation of exogenous DNA or RNA of an editing system, and reduces potential safety risks caused by addition of exogenous genetic materials. In addition, the added PJ34 is good in compatibility with an existing editing system and can be combined with other optimization strategies for use, and therefore the pilot editing efficiency is further improved. In conclusion, a convenient and efficient way is provided for improving the editing efficiency of the pilot editing, and a basis is provided for wide application of the pilot editing.

Provided are a gene editing method for the simultaneous knock-in of DNA fragments at multiple sites and an application thereof. The method comprises the following steps: making a plurality of double-stranded DNA nicks in a target gene; designing a plurality of homology-directed repair templates; designing exogenous DNA fragments on the homology-directed repair templates; constructing the plurality of homology-directed repair (HDR) templates on the same vector in series; and respectively enabling knock-in of the exogenous DNA fragments into the double-stranded DNA nicks by means of the vector. The multi-site multi-fragment gene knock-in provided by this gene editing method can bring a plurality of DNA fragments into a single vector by means of HDR and enable simultaneous and precise insertion of a plurality of exogenous genes into a target genome at distinct sites, thereby greatly reducing gene editing costs and also increasing the editing efficiency. The gene editing method can be used for basic research and clinical application research.