62 results about "Exogenous DNA" 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.

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 synthetic biology genome editing, and relates to a chassis strain for gene cluster in-vivo traceless editing, a construction method and a gene cluster traceless editing method.The chassis strain is based on escherichia coli BL21 (DE3) and integrates a CRISPR-Cas9 system and a homologous recombination system (lambda Red) of a lambda bacteriophage source; and then knocking in an inducible sgRNA expression cassette (sgT) of a targeted editing vector to construct the vector. The strain has an efficient editing capability aiming at exogenous DNA, traceless editing of a target biosynthetic gene cluster is realized, and editing plasmids can be quickly eliminated, so that iterative editing is facilitated. The method is simple, convenient and rapid to operate and high in practicability, and provides powerful technical support for metabolic engineering and synthetic biology breeding transformation of streptomyces industrial strains, activation of synthetic gene clusters of recessive natural products and excavation of novel natural products.

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.

Disclosed is a detection method for a dominant clone caused by an exogenous DNA insertion mutation, comprising: carrying out pretreatment, including plus-minus strand correction and redundancy integration, on virus IS information data; step 2, when the pretreatment is completed, identifying ISs caused by non-specific amplification so as to remove a false positive IS detection result caused by PCR non-specific amplification; step 3, merging the ISs at neighboring positions on the basis of a site clustering algorithm, and clustering the ISs; and step 4, analyzing a clone plane on the basis of the clustering result, and describing a dominant clone. According to the present invention, a PCR primer similarity ranking algorithm is utilized to identify and eliminate non-specific amplification potentially caused by PCR primer similarity, significantly reducing false positives of IS identification in practical applications, improving the accuracy.

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.

A method for genome editing in isolated cells, characterized in that an exogenous DNA with a homologous arm having a length of less than 500 bp is introduced into a target genome by performing homologous recombination on at least one of the 5'-end and 3'-end of the exogenous DNA.

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

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 present disclosure provides a hotspot useful for transformation of animal cells by TI (Targeted Integration). The hotspot of the present disclosure was found near LOC103164262 in the CHO cell genome. Alternatively, the present disclosure relates to transformed cells into which an exogenous DNA has been introduced into said hotspot, and a method of producing a polypeptide encoded by the DNA by culturing the cells.

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.

The invention discloses an automatic process for pichia pastoris microplate-level batch gene editing. According to the method, experimental steps and parameter details of pichia pastoris chemical conversion are optimized in a targeted manner, and a pichia pastoris chemical conversion total reaction system is compressed to be adaptive to automatic high-throughput pore plate operation; the adjustment range includes but is not limited to competent preparation liquid components and proportion, conversion mixed liquid component composition and proportion, heat shock temperature, heat shock duration, ssDNA and exogenous DNA addition amount and proportion and the like. High-throughput competent preparation and batch chemical conversion of free plasmids are realized in pichia pastoris by using a synthetic biological automation device. The CRISPR technology is combined, batch single-gene editing and batch double-gene editing of pichia pastoris are achieved through an automatic program, and the integration efficiency reaches 81.4% and 36.1%. The automatic experiment process can be used for multi-gene iteration transformation of other unconventional yeasts such as pichia pastoris and the like, and can be suitable for industrial application.

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.

A double-stranded DNA adapter and a detection method for short DNA fragments, relating to the technical field of molecular biological detection. The double-stranded DNA adapter comprises: a first adapter as shown in SEQ ID NO: 1, and a second adapter as shown in SEQ ID NO: 2. The provided detection method has high detection accuracy, is time-saving and simple, has a low detection limit, and has universality in detection application. The provided double-stranded DNA adapter has the technical advantage of a wide detection range for short DNA fragments, and has a good detection effect for any exogenous DNA. For detecting exogenous residual DNA, there is no need to design specific primers for specific DNA samples.

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 discloses a recombinant filamentous fungus transformant screening method based on Zhongshengmycin resistance. The Zhongshengmycin is used as a pressure source for screening filamentous fungus gene modification transformants, and the nat gene is used as a Zhongshengmycin resistance gene, so that exogenous DNA can be efficiently integrated into a filamentous fungus genome with low background. According to Zhongshengmycin resistance-based transformant screening, the transformation efficiency is not lower than that of common markers, and the genetic background is far lower than that of common antibiotics of filamentous fungi such as hygromycin. An efficient selection marker is added for rational transformation of filamentous fungi, multiple antibiotics including zhongshengmycin, hygromycin and the like can be used in the same wild type strain for multiple screening, the gene continuous transformation difficulty of the strain is greatly reduced, the continuous genetic transformation mode of a filamentous fungi synthetic biological chassis is enriched, and the application prospect is wide. The method expands thinking for construction of industrial filamentous fungus microbial cell factories, and has important reference value for high-throughput screening of other filamentous fungi.

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.