1534 results about "Genome editing" patented technology

The invention belongs to the technical field of gene engineering, and particularly relates to mutants of murA and ygeA genes and application of the mutants in production of human milk oligosaccharides. According to the invention, murA and ygeA genes of escherichia coli are edited by using a gene editing technology, so that a genetic engineering chassis strain carrying murA and ygeA mutants is obtained. Further, the chassis strain is applied to the construction process of production strains of LNT II, LNnT, LNT, 3 '-SL and 6'-SL, and the yield of the LNT II, the LNnT, the LNT, the 3 '-SL and the 6'-SL is further increased.

The invention provides a construction method and application of a pichia pastoris strain capable of efficiently secreting and expressing recombinant human-derived protein, and relates to the technical field of synthetic biology. According to the invention, pichia pastoris is used as a chassis strain, a ku70 gene and / or a pep4 gene in a pichia pastoris genome are / is knocked out through a gene editing technology, and a new signal peptide O factor signal is introduced, so that the secretion efficiency of the pichia pastoris for secreting and expressing recombinant human-derived protein can be remarkably improved, the problem of protein degradation is effectively improved, and the recombinant human-derived protein can be obtained. And meanwhile, a high-copy pichia pastoris strain is efficiently obtained, so that the expression quantity of recombinant human protein is improved, and powerful support is provided for research and development of related drugs and biological manufacturing.

The present invention discloses a Gs12-7 endonuclease variant and a gene editing system mediated thereby. Specifically, by means of a rational mutation strategy, two Gs12-7 mutants were constructed and compared, and it was found that after the 157th amino acid of the Gs12-7 endonuclease was mutated from Glu to Arg, the activity of this gene editing enzyme could be significantly improved, and this variant was designated as Gs12-7MAX. The present invention provides an efficient gene editing technology mediated by the CRISPR-Gs12-7MAX system, which has broad application prospects in the field of genome site-directed modification.

Provided are compositions for genome editing and site-directed integration in plants comprising microprojectile particles coated, treated of applied with a recombinant DNA construct comprising a sequence encoding one or more genome editing reagents for delivery to a mature embryo explant from dry seeds. Further provided are methods for genome editing and site-directed integration in at least one cell of a plant using the disclosed compositions, and plants, plant parts and seeds comprising an edited genome or site-directed integration, which are produced by the disclosed methods.

The invention provides a keto-alcohol acid reductoisomerase mutant and application of the keto-alcohol acid reductoisomerase mutant in construction of genetically engineered bacteria for high yield of D-pantothenic acid. In order to solve the technical problem that in the prior art, the yield of D-pantothenic acid synthesized through microbial fermentation is not high, semi-rational design of enzyme is adopted, and a keto-alcohol acid reductoisomerase mutant with substrate specificity is obtained and applied to construction of genetically engineered bacteria for high yield of D-pantothenic acid. According to the invention, a genetically engineered bacterium with high yield of D-pantothenic acid is constructed by utilizing the keto-alcohol acid reductoisomerase mutant through a CRISPR-Cas9 gene editing technology, so that the yield of D-pantothenic acid is increased by about 12.04%, and compared with an original strain, the valine accumulation amount in obtained fermentation liquor is almost unchanged compared with that of a control, and the yield of D-pantothenic acid is greatly improved. However, the accumulation of branched chain amino acid isoleucine is less, so that the modification of the substrate specificity of the keto-alcohol acid reductoisomerase is achieved.

The specification provides methods for introducing a desired genetic change in a nucleotide sequence using a double-strand break (DSB)-inducing genome editing system, the method comprising: identifying one or more available cut sites in a nucleotide sequence; analyzing the nucleotide sequence and available cut sites with a computational model to identify the optimal cut site for introducing the desired genetic change into the nucleotide sequence; and contacting the nucleotide sequence with a DSB-inducing genome editing system, thereby introducing the desired genetic change in the nucleotide sequence at the cut site.

The invention discloses an optimized NlovFz2-omega RNA (Ribonucleic Acid) editing system, the optimal NlovFz2 system enNlovFz2 is obtained by modifying NlovFz2 protein and an omega RNA skeleton, and on the basis of remarkably improving the editing efficiency compared with a parent NlovFz2 system, the NlovFz2 mutant protein and T5 exonuclease are fused to obtain an enNlovFz2-T5 system with higher editing efficiency. The method has important significance and value for promoting wide application of a gene editing technology in the aspects of functional mechanism research and clinical treatment.

The invention discloses application of an RLCK86 gene in improvement of drought tolerance of tomatoes, and belongs to the technical field of biology. According to the application, the tomato RLCK86 gene function is silenced or deleted by utilizing a biological technical means, so that the tolerance of tomato plants to drought stress is improved; the nucleotide sequence of a protein coding region of the RLCK86 gene is as shown in SEQ ID NO. 1. The invention discloses the application of the RLCK86 gene in regulating and controlling the drought stress resistance of the tomato for the first time, the function of the RLCK86 gene is deleted through a gene editing technology, the drought tolerance of the tomato can be remarkably improved, and a reference is provided for cultivating drought-resistant tomato germplasm resources.

The invention belongs to the technical field of gene editing, and particularly relates to a poa pratensis gene editing method. The invention provides a bluegrass CRISPR / Cas9 gene editing system, which comprises a pCAMBIA1300-Cas9 vector, a bluegrass gene PpTCP4 target sequence and a primer pair for amplifying the bluegrass gene PpTCP4 target sequence, the gene PpTCP4 of the bluegrass is directionally edited by utilizing a CRISPR / Cas9 gene editing technology, gene editing of a base mutation type is realized, a gene editing plant is successfully obtained, and the gene editing effect is good. The gene editing efficiency is 0.67%, and new research basis and technical reference are provided for developing the directional improvement of the poa pratensis to create high-quality germplasm resources.

The present disclosure is directed to controlling pest infestation by inhibiting one or more biological functions in an invertebrate pest. The disclosure discloses methods and compositions for use in controlling pest infestation by feeding one or more different recombinant double stranded RNA molecules to the pest in order to achieve a reduction in pest infestation through suppression of gene expression. The disclosure also discloses methods and compositions for targeted genome editing in the pest in order to achieve a reduction in pest infestation through disruption of protein activity. The disclosure is also directed to methods for making transgenic plants that express the double stranded RNA molecules and targeted genome editing constructs for use in protecting plants from pest infestation.

The invention belongs to the technical field of gene engineering, and particularly relates to an RNA polymerase beta subunit mutant and application thereof. The RpoB mutant is obtained through a gene editing technology, after the 560th amino acid of the RpoB is changed into leucine from proline, the number of living cells during strain fermentation is greatly increased, the tolerance of the RpoB mutant in the later environment of a fermentation tank is improved, the RpoB mutant is applied to escherichia coli for producing human milk oligosaccharide, and finally the yield of the human milk oligosaccharide is greatly increased.

The invention discloses application of a substance with a TaSWEET11f gene knocked out to improvement of gibberellic disease resistance of wheat crops, and relates to the technical field of agricultural biology. According to the application, the correlation between the TaSWEET11f gene and the gibberellic disease resistance is found for the first time, three homologous genes TaSWEET11f-A, TaSWEET11f-B and TaSWEET11f-D of the gene are obtained through screening, the wheat TaSWEET11f gene is specifically knocked out in a receptor material by adopting a CRISPR / Cas9 gene editing technology, gibberellic disease resistance identification is carried out by utilizing a single-flower instillation method, and a result shows that compared with a receptor material Fielder, the wheat TaSWEET11f gene has the advantages that the wheat TaSWEET11f gene can be compared with the receptor material Fielder; the gibberellic disease resistance of a plant edited by the CR-Tasweet11f gene is obviously improved; according to the invention, the wheat TaSWEET11f gene is knocked out by using a CRISPR / Cas9 technology, so that a technical support is expected to be provided for directionally and rapidly creating new germplasm resistant to gibberellic disease, and the method has wide breeding utilization value.

The invention discloses an application of a soybean nucleolus GTP binding protein gene GmNSN1 in soybean resistance to soybean mosaic virus diseases. The invention relates to an application of a soybean nucleolus GTP binding protein gene GmNSN1 as shown in SEQ ID NO.1 and SEQ ID NO.2 in genetic engineering modification of soybean mosaic virus resistance. The soybean nucleolus GTP binding protein gene GmNSN1 can negatively regulate and control the resistance of soybeans to diseased soybean mosaic virus diseases, and the antiviral response of the soybeans can be activated due to function deficiency of the soybean nucleolus GTP binding protein gene GmNSN1. The gene is knocked out through a CRISPR / Cas9 gene editing technology, so that the resistance of soybeans to the soybean mosaic virus disease can be remarkably improved.

The invention relates to a gene editing vector of agrobacterium, a gene editing method of the gene editing vector and an agrobacterium editing strain. The agrobacterium gene editing vector comprises the following gene elements: a plasmid skeleton, a Pvan promoter, a spCas9 gene, a green fluorescent protein gene GFP, gRNA, and an upstream homologous arm and a downstream homologous arm of a target gene. According to the agrobacterium gene editing vector, a CRISPR / spCas9-GFP gene editing system is adopted, an efficient, simple, convenient and traceless genome editing system is constructed in agrobacterium, and gene knockout and insertion can be achieved.

The invention discloses a toxoplasma gondii attenuated vaccine strain RH delta rop67 as well as a construction method and application thereof, and belongs to the technical field of parasitic disease prevention and control and biological product preparation. The attenuated vaccine strain is constructed by performing targeted knockout on the ROP67 gene in a toxoplasma gondii strain RH delta ku80 through a CRISPR / Cas9 mediated gene editing technology. Compared with a wild type strain, the attenuated vaccine strain shows remarkable attenuation characteristic and good immunogenicity. A test result shows that the attenuated vaccine strain can induce a host to generate specific immune response mainly based on cellular immunity, maintains a protection effect on toxoplasma gondii infection in a relatively long immune period, and has a protection effect on tachyzoite infection and a chronic infection stage of toxoplasma gondii strains with different virulence; the survival ability of a host to tachyzoite infection can be improved, and the formation level of cysts in tissues is reduced. The invention provides a technical scheme with long-term immune potential for research and development of toxoplasma gondii attenuated vaccines.

The invention discloses a rice salt stress resistant gene OsHAK11 coding protein and application thereof, and belongs to the field of plant genetic engineering. According to the method, a rice OsHAK11 gene (the nucleotide sequence is shown as SEQ ID NO.1) is knocked out through a CRISPR / Cas9 gene editing technology, and a mutant with significantly enhanced salt tolerance is obtained. The gene editing vector pEGCas9Pubi-B-OsHAK11 contains sgRNA of a region as shown in a target SEQ ID NO.4, a rice receptor material is transformed through agrobacterium tumefaciens mediation, and a plant with the OsHAK11 gene subjected to frame shift mutation is obtained through screening. A salt stress experiment shows that the survival rate of the mutant oshak11 is obviously higher than that of a wild type. The invention provides a new gene resource and an efficient technical means for salt-tolerant breeding of rice.

The invention provides a keto-alcohol acid reductoisomerase mutant and application of the keto-alcohol acid reductoisomerase mutant in construction of genetically engineered bacteria for high yield of D-pantothenic acid. In order to solve the technical problem that in the prior art, the yield of D-pantothenic acid synthesized through microbial fermentation is not high, semi-rational design of enzyme is adopted, and a keto-alcohol acid reductoisomerase mutant with substrate specificity is obtained and applied to construction of genetically engineered bacteria for high yield of D-pantothenic acid. According to the invention, a genetically engineered bacterium with high yield of D-pantothenic acid is constructed by utilizing the keto-alcohol acid reductoisomerase mutant through a CRISPR-Cas9 gene editing technology, so that the yield of D-pantothenic acid is increased by about 12.04%, and compared with an original strain, the valine accumulation amount in obtained fermentation liquor is almost unchanged compared with that of a control, and the yield of D-pantothenic acid is greatly improved. However, the accumulation of branched chain amino acid isoleucine is less, so that the modification of the substrate specificity of the keto-alcohol acid reductoisomerase is achieved.

The invention discloses an application of a soybean ARGONAUTE family gene GmAGO5 in soybean resistance to soybean mosaic virus diseases. The invention also discloses application of the soybean ARGONAUTE family gene GmAGO5 as shown in SEQ ID NO.1 and SEQ ID NO.2 in genetic engineering modification of soybean mosaic virus resistance. The soybean ARGONAUTE family gene GmAGO5 can negatively regulate and control the resistance of soybeans to diseased soybean mosaic virus diseases, and the antiviral response of the soybeans can be activated due to function deficiency of the soybean ARGONAUTE family gene GmAGO5. The gene is knocked out through a CRISPR / Cas9 gene editing technology, so that the resistance of soybeans to the soybean mosaic virus disease can be remarkably improved.

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a rice cold tolerance negative regulation gene OsbHLH090 and application thereof. The nucleotide sequence of the gene is shown as a sequence table SEQ ID NO: 1, and the coded amino acid sequence is shown as a sequence table SEQ ID NO: 2. According to the invention, the rice OsbHLH090 gene is edited by using a CRISPR-Cas9 gene editing technology, and the gene is knocked out to obtain the cold-sensitive rice mutant. The method comprises the following steps: cloning OsbHLH090CDS into a pC1390U vector, and constructing an overexpression vector; under cold stress, the survival rate of the mutant is higher than that of a wild type, and the survival rate of overexpressed transgenic rice is lower than that of the wild type, so that the OsbHLH090 gene is an important negative regulation factor in a rice cold-tolerant approach. The invention provides a new resource for cold-resistant breeding of plants and rice.

The invention belongs to the technical field of gene editing, and discloses a gene editing system and application. The invention provides a gene editing system. The gene editing system comprises Cas9 nickase, sgRNA (small guide ribonucleic acid) and annular petRNA. According to the present invention, the target chain cutting mediated reverse pilot editing (Split Reverse Prime Editing, srPE) realizes the precise editing of the non-target chain cutting site upstream sequence, such that the gene editing coverage range is expanded from the specific region only limited to the non-target chain cutting site downstream to the whole genome range, the stable and efficient editing ability is maintained in different cell types, and the target chain cutting mediated reverse pilot editing can be provided for the target chain cutting site. And meanwhile, the off-target risk is reduced. The srPE gene editing technology breaks through the fundamental limitation of the prior art, and provides a more comprehensive and more efficient technical tool for precise medicine and gene therapy.

The invention discloses application of an upstream non-coding region uORF of an ABA2 gene in regulation and control of crop agronomic traits, in particular to application in improvement of pre-harvest sprouting resistance of rice and cultivation of pre-harvest sprouting resistant rice strains, and belongs to the technical field of gene engineering. The invention finds that uORF in the upstream non-coding region of the crop ABA2 gene can inhibit the translation level of downstream ABA2 protein on the basis of not influencing the transcriptional level, so that the uORF can participate in regulation and control of crop agronomic traits in which ABA participates in regulation and control, such as improvement of pre-harvest sprouting resistance of rice. Therefore, the upstream non-coding region uORF of the crop ABA2 gene can be modified through a hybridization or gene editing technology, so that the purpose of regulating and controlling crop agronomic traits in which ABA participates in regulation and control is achieved, for example, site-directed mutagenesis is performed on an initiation codon of the upstream non-coding region uORF of the rice ABA2 gene, and the pre-harvest sprouting resistance of rice is improved.

The invention discloses a construction method and application of an NMD escape model of an Alport syndrome mouse. A non-human animal model carrying Col4a5 gene c.4432delG frame shift mutation is prepared on the basis of a CRISPR / Cas9 gene editing technology. The method comprises the following steps: co-injecting gRNA of a 49 exon of a targeted Col4a5 gene, homologous recombinant donor oligonucleotide containing c.4432delG mutation and Cas9 nuclease into a mouse fertilized egg, and carrying out embryo transplantation to obtain an F0-generation mutant mouse; a mutation site is verified by combining PCR (Polymerase Chain Reaction) with sequencing, and a stably inherited mutation line is established through two generations of breeding. Through verification, the model accords with pathological characteristics of the Alport syndrome, can stably simulate typical clinical manifestation and pathological characteristics of the human X-linked Alport syndrome, and can be used as an important tool for research of the Alport syndrome.

The invention belongs to the field of citrus breeding, and discloses application of a citrus CsERF113 gene in regulation and control of plant fruit abscission. It is found for the first time that the CsERF113 gene can induce plant separation layer formation and fruit abscission, which has a great promotion effect on molecular mechanism research of abscission regulation of plants including organs such as fruits and the like; the citrus CsERF113 gene cloned by the invention has a remarkable induction effect on citrus fruit abscission, which is of great significance to cultivation of overwintering non-abscission late-maturing citrus varieties through a gene editing technology.

The invention discloses application of programmable TnpB nuclease gene editing, and relates to the technical field of gene editing. The TnpB nuclease is any one of (A1), (A2) and (A3): (A1) TnpB nuclease, the amino acid sequence of which is as shown in SEQ ID NO.1; (A2) carrying out substitution, deletion and / or addition of one or more amino acid residues on the amino acid sequence of the TnpB nuclease to obtain a protein which has 80% or more of identity with the TnpB nuclease and has the same biological function with the TnpB nuclease; and (A3) a fusion protein obtained by connecting a protein tag to the N terminal or / and C terminal of (A1) or (A2). The TnpB nuclease provided by the invention has cis-cleavage activity and trans-cleavage activity, and has a wide application prospect in the fields of nucleic acid detection and gene editing.

The invention belongs to the technical field of gene engineering, and particularly relates to an LgtA mutant and application thereof in production of human milk oligosaccharide. According to the present invention, a gene editing technology is adopted to mutate a key enzyme beta-1, 3-N-acetyl glucosamine transferase for synthesizing an intermediate product LNT II so as to obtain a mutant having at least one of R13H mutation, L24M mutation and R205C mutation on the basis of a wild type LgtA represented by SEQ ID NO.1; further, the mutant is applied to the construction process of production plasmids of the LNTII, the LNnT and the LNT, and the yield of the LNTII, the LNnT and the LNT is further increased.

The present invention provides a chimeric nucleic acid-guided nuclease including at least one domain substituted with distinct bacterial species. These chimeric nucleases improve function of modified guide polynucleotides, enhancing the genomic modification abilities of nucleic acid-guided nucleases. CRISPR-Cas systems such as those described herein are often used in genome editing, but the complexity of the polypeptides and polynucleotides leads to unpredictable outcomes. The chimeric CRISPR-Cas system disclosed herein was surprisingly able to rescue function of a previously non-functional guide nucleotide. The nucleases and methods described herein are useful for treating diseases associated with a gene or polynucleotide.

The invention provides a method for constructing a rice epigenetic recombinant inbred line, and belongs to the technical field of molecular biology and genetic breeding. According to the method for constructing the rice epigenetic recombination inbred line, a fertile rice DDM1 gene function weakening mutant is obtained through a CRISPR / Cas9 gene editing technology and hybridized with wild type rice with the same background to obtain F1, then the F1 and the wild type rice are subjected to backcrossing to obtain BC1F1, single-plant offspring selfing with the DDM1 genotype being the wild type is selected, selfing is conducted for multiple generations through a single-grain transmission method, and the rice epigenetic recombination inbred line is obtained. According to the present invention, the strain population with stable inheritance is constructed, such that the rice epigenetic recombinant inbred line is obtained, and has characteristics of rich phenotype, genetic and epigenetic variation so as to provide important population resources for batch identification and the like of important agronomic shape functional sites of rice.

The invention belongs to the technical field of gene editing, and discloses a base editor system and application. According to the invention, a heterologous peptide sequence is inserted into IscB protein, the obtained peptide embedded IscB is fused with deaminase or glycosylase to construct a base editor, and DNA base editing is carried out. According to the base editor system, the size limitation is overcome, and single AAV delivery is achieved; the editing efficiency is obviously improved; the broad-spectrum applicability is realized; the system optimization is more flexible; the method has efficient editing potential in vivo, and the off-target effect is reduced. The invention provides an important new strategy for hyperlipemia treatment and provides a basis for gene therapy of other hereditary diseases.

The invention relates to the technical field of gene editing, and particularly provides a method for creating herbicide-resistant rice through gene editing of an OsEPSPS gene promoter region, and the method is characterized in that WT / 29bp single allelic deletion mutation is introduced into the OsEPSPS gene promoter region, i.e., A in an OsEPSPS gene initiation codon ATG is 0 site, A in the OsEPSPS gene initiation codon ATG is 0 site, A in the OsEPSPS gene initiation codon ATG is 0 site, and A in the OsEPSPS gene initiation codon ATG is 0 site; 29bp is deleted from the 112nd site to the 1150th site at the upstream of the initiation codon ATG on one allele, and the other allele is kept unchanged. By introducing the single allelic deletion mutation, the rice can obtain resistance to EPSPS inhibitor herbicides, the resistance is identified in T0-generation plants carrying the mutation and can be stably inherited to offspring plants, and the application value is high for guaranteeing agricultural production safety and improving the use efficiency of the herbicides.

The invention provides an application of WNT7B in construction of a myopia animal model. Homologous genes wnt7ba and wnt7bb of the WNT7B in the zebra fish are knocked out or knocked down by applying a CRISPR / Cas9 gene editing technology, an animal model of which the eye axis length is remarkably increased, the eyeball movement frequency is remarkably reduced and the light response is reduced is obtained, and the result shows that the WNT7B is related to the high myopia. The model provides a favorable tool for pathogenesis and drug screening of high myopia, and has a good application prospect.