1989 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.

The present disclosure provides compositions and methods for the selective transduction and genome editing of human cells (e.g., hematopoietic stem and progenitors cells, HSPCs) using engineered viral like particles (eVLPs). Aspects of the disclosure provide eVLP compositions comprising fusion proteins comprising a targeting moiety. In some embodiments, the fusion proteins comprise a cytokine conjugated to a transmembrane protein and / or an envelope glycoprotein. In other embodiments, the fusion proteins comprise a targeting moiety domain, a stalk protein domain, a transmembrane and / or envelope glycoprotein domain. Targeted-eVLP architectures comprising various targeting domains, stalk domains, transmembrane domains, and envelope glycoproteins are also provided herein. Other aspects of the disclosure provide eVLP compositions comprising envelope glycoproteins comprising non-natural sugars and methods of conjugating said eVLPs to various targeting moieties using bio-orthogonal click chemistry. Polynucleotides, vectors, cells, and kits useful for producing the articles, and performing the methods, described herein are also provided.

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 relates to the field of nucleic acid editing, in particular to the technical field of regularly clustered interval short palindromic repeat (CRISPR). In particular, the present invention relates to Cas effector proteins, fusion proteins comprising such proteins, and nucleic acid molecules encoding them. The invention also relates to complexes and compositions for nucleic acid editing (e.g., gene or genome editing) comprising the proteins or fusion proteins of the invention, or nucleic acid molecules encoding them. The invention also relates to methods for nucleic acid editing (e.g., gene or genome editing) using the proteins or fusion proteins comprising the invention.

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 discloses an OpenCRISPR (Open Clustered Regularly Interspaced Short Palindromic Repeats)-NLS (Non-Least Squares) fusion protein for gene editing of penaeus monodon, a construction method of the OpenCRISPR-NLS fusion protein and a nucleotide construct containing the OpenCRISPR The invention also discloses an application of the fusion protein or the nucleotide construct in gene editing of penaeus monodon and an application of the fusion protein or the nucleotide construct in population improvement of aquaculture penaeus monodon. According to the invention, codon optimization is carried out on a coding sequence of OpenCRISPR-1 protein, and a nuclear localization signal (NLS) is fused with the OpenCRISPR protein subjected to codon optimization; the method has the advantages of being efficient in gene expression, accurate in targeting positioning capacity, excellent in genome editing efficiency, capable of reducing the off-target effect, wide in adaptability, capable of promoting sustainable aquaculture, remarkable in economic and social value and the like.

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 provides a fusion protein with sequence preference and a gene editing system, and relates to the technical field of gene editing. The invention discloses that the key 48th amino acid for binding TadA-8e and substrate DNA influences the overall editing efficiency, editing accuracy and sequence preference of ABE8e for the first time; through rational design, a TadA-8e part in ABE8e is modified, saturation mutation is designed at a key 48th amino acid for combining the TadA-8e and a substrate DNA, and YAY high-precision ABE with sequence preference is developed. According to the method, A in a sequence YAY (Y = T or C) in an editing window can be accurately edited into G, other A is not edited or only low editing is generated, the problem of'adjacent base 'mutation is avoided, and potential safety hazards caused by wrong mutation are reduced.

The invention discloses a method for constructing a Ddx58-R109C point mutation mouse model by using a CRISPR / Cas9 gene editing technology and an application of the Ddx58-R109C point mutation mouse model. The method comprises the following steps: (1) target construction: searching a CDS region of a Ddx58 gene, determining an exon part, and determining a gene mutation site; (2) obtaining a fertilized egg, and injecting Cas9mRNA, gRNA and donor oligo into the fertilized egg of the C57BL / 6J mouse together in vitro through a microinjection technology to obtain an F0-generation hybrid mouse; (3) mating the F0-generation hybrid mouse with the wild type mouse to obtain an F1-generation mouse; (4) the F1-generation heterozygous mice which are identified to be positive are subjected to mating, and homozygous Ddx58-R109C mutant type and wild type mice can be obtained by breeding obtained offspring; (5) phenotypic analysis shows that Ddx58-R109C point mutation mice can have pathological changes of systemic lupus erythematosus and lupus nephritis; the invention can provide an effective research approach and method for researchers to deeply explore the action mechanism of Ddx58-R109C mutation in diseases, and has important significance for explaining the pathogenesis of systemic lupus erythematosus and researching and developing targeted therapeutic drugs.

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.

Provided herein are compositions and methods for modification of the retron editing system to enable multiple simultaneous edits in prokaryotic and / or eukaryotic cells. Such compositions and methods find use in, for example, therapeutic editing and cell engineering, such as for bioproduction.

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 relates to a palm crop protoplast gene editing method based on a CRISPR / Cas9 system and application of the palm crop protoplast gene editing method, and belongs to the technical field of biology. A carrier containing PDS gene editing targets and Cas9 protein genes of palm crops is successfully delivered to protoplasts of the palm crops such as coconuts and areca nuts through protoplast transformation for the first time, and the carrier contains sgRNA sequences designed according to PAM sequences recognized by Cas9 protein. The sgRNA and Cas9 compound formed after expression in palm crop protoplast cells can be edited at a PDS gene editing target. According to the method disclosed by the invention, an editing carrier based on the CRISPR / Cas system can be delivered into the palm crop protoplast, the method is used for gene editing technology research of palm crops such as coconuts, betel nuts, oil palms, date palms and the like, and the feasibility of the method for editing the palm crop protoplast genome target gene based on the CRISPR / Cas 9 system is verified; and a rapid and effective method is provided for subsequently establishing an efficient palm crop protoplast gene editing technical system.

The invention relates to a construction method of a macrophage specific fluorescence report mouse model, and belongs to the technical field of genetic engineering and genetic modification. According to the invention, a brand-new C1qc-CreChy gene knock-in mouse model is obtained after a C1qc gene locus is edited through a CRISPR / Cas9 genome editing technology for the first time, the expression of Cre and mCherry is directly driven by a C1qc promoter, and flow cytometry verifies that mCherry has very high expression quantity only in microglial cells in the brain of a mouse and macrophages in the spleen of the mouse. And mCherry expression is not detected in other immune cells, so that the two macrophages are traced and used for researching the functions of the macrophages. The invention provides a usable animal model for researching heterogeneity of macrophages, and provides a new animal model for researching complex immune functions in a body.

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 belongs to the technical field of gene engineering, and particularly discloses a construction method and application of a toxoplasma gondii gene knockout vaccine strain, a TGGT1202920 gene of toxoplasma gondii is directly knocked out through a gene editing technology to obtain a delta TGGT1202920 strain, and the strain has the characteristics that the strain can be normally cultured in vitro and is almost non-toxic in vivo; after animals are immunized with delta TGGT1202920 strains, good immune protection can be generated, infection of wild type toxoplasma gondii can be resisted, and the strain can be used for preparing toxoplasmosis resistant vaccines to prevent toxoplasma gondii infection.

The invention belongs to the technical field of genetic engineering and molecular breeding, and particularly relates to application of TaHPPD protein in regulation and control of wheat stripe rust resistance. The TaHPPD protein disclosed by the invention can be used for weakening the accumulation of reactive oxygen species (ROS) and cell necrosis reaction caused by CNL protein NLRMro and reducing ion leakage. A TaHPPD gene in wheat is knocked out through a gene editing technology, a TaHPPD knockout (TaHPPD-KO) wheat material is obtained, and wheat with wheat stripe rust resistance is obtained. The result of the embodiment shows that the TaHPPD-KO wheat plant shows obvious resistance compared with the control Fielder. The TaHPPD protein can be used for regulating and controlling the stripe rust resistance of wheat, and can be used for cultivating stripe rust resistant wheat.