29 results about "Genomic engineering" patented technology

Systems and methods for highly-effective CRISPR-Cas based genomic editing within human chromosome 15q11-q13 have been developed as therapeutic interventions for Angelman Syndrome (AS). Selective single guide RNA molecules (sgRNAs) that impart enhanced CRISPR-Cas editing of genomic mutations associated with Angelman Syndrome in human cells are described. The engineered sgRNAs induce activity of non-pathogenic, paternal UBE3A alleles to reduce, reverse and / or prevent the causative neurodevelopmental defects of AS in a subject in need thereof. Compositions and methods of engineered crRNAs, sgRNAs thereof and ribonucleoprotein (RNP) complexes thereof are provided for enhanced genomic engineering with increased on-off target specificity and on-target editing efficacy for treatment of AS.

The invention belongs to the field of gene engineering and enzyme engineering, and particularly relates to mannase derived from space mutagenesis yeast strains as well as a preparation method, a product and application of the mannase. The amino acid sequence of the mannase is as shown in SEQ ID NO.2, and the nucleotide sequence of the mannase is as shown in SEQ ID NO.3. The mutant retains 70% of enzyme activity at a high temperature (85 DEG C for 3 min, and only 40% of original enzyme remains at 85 DEG C for 3 min) and has an acid pH value (the stability is improved from 3% to 25% after treatment for 180 min at pH 3.0), and meanwhile, the mutant has the low-temperature efficient catalytic characteristics that the optimum pH value is 7.0, the optimum temperature is 40 DEG C, and 30% of enzyme activity is kept at 20 DEG C, and has remarkable application potential in the fields of feed, paper-making industry and the like.

This invention discloses a high-activity, heat-resistant α-glucan phosphorylase mutant and its applications, belonging to the fields of genetic engineering and modern enzyme technology engineering. This invention utilizes site-directed mutagenesis technology to modify the wild-type... α‑ Dextran phosphorylase was modified to construct a series of mutants. Compared with the wild-type enzyme, the enzyme activity was increased by 1.68 to 2.49 times, and the mutants had a wider range of adaptability to temperature and pH. Among them, the mutant R282Y / H506A showed excellent thermostability. After incubation at 60℃ for 132 h, its enzyme activity was still 50.96%, and after incubation at 70℃ for 60 h, its enzyme activity was still 53.71%. The significantly improved thermostability creates better conditions for the use of this enzyme in practical applications.

The invention discloses a glycosyl transferase mutant for synthesizing rebaudioside M as well as a product and application thereof, and belongs to the technical field of gene engineering. The technical problem to be solved is that the existing glycosyl transferase and sucrose synthase are low in enzyme activity and insufficient in enzyme activity stability, so that the synthesis efficiency of rebaudioside M (RebM) is not high, and the industrial application of preparing RebM by an enzyme method is limited. According to the key points of the technical scheme, the glycosyl transferase mutant is provided, the mutant is obtained by mutating UDP-glycosyl transferase UGT76G1, and the amino acid sequence of the UDP-glycosyl transferase UGT76G is as shown in SEQ ID NO. 1; the mutation comprises mutation on the 83rd site of SEQ ID NO.1, and the amino acid sequence of the glycosyl transferase mutant is shown as SEQ ID NO.5. The invention further discloses a preparation method of the glycosyl transferase mutant.

The present invention relates to the field of genomic engineering. In particular, a chimeric prime editing (cPE) system is disclosed comprising elements including, but not limited to a Cas9 nickase (nCas9) / high fidelity nucleotide polymerase (HFNTPol) RNA, one or more single guide RNAs (sgRNAs), and a chimeric prime editor template oligonucleotide (cpetODN) comprising a deoxyribonucleic acid nucleotide polymerase template (NPT) and a primer binding site. For example, the sgRNA and the cpetODN are ligated into a single oligonucleotide. Alternatively, the sgRNA and the cpetODN are free and independent molecules (e.g., modular). This cPE system results in precise and efficient genome editing in cells and in adult mouse liver which is advantageous over conventional sgRNA prime editor fusion constructs. This flexible and modular system is an improvement in the art to obtain precise genome editing.

The application belongs to the technical field of genetic engineering, and discloses a high-efficiency micro CRISPR / Cas9 gene editing system and application. The amino acid sequence of the Cas9 protein is shown as SEQ ID NO. 1, or is a sequence in which at least one amino acid in the amino acid sequence shown as SEQ ID NO. 1 is replaced, deleted or inserted. The Cas9 protein of the application can maximize the simplification of the protein structure while maintaining the core catalytic activity, can improve the editing efficiency in different application scenarios, can effectively target the genomic region where the non-standard PAM sequence is located, and can improve the editing capacity of the CRISPR system in the complex genome. The application provides a new idea for the diversified development of gene editing technology.

The invention relates to the technical field of gene engineering technology, in particular to a preparation method of an efficient and stable gene knockout cell line. According to the AKAP11 gene knockout cell line provided by the invention, the 6th exon of the AKAP11 gene in the cell line is subjected to 3395bp frameshift deletion mutation, so that a coding frame is shifted, and protein translation is terminated in advance; after continuous subculture, the homozygous knockout phenotype is still maintained through PCR sequencing verification. A CRISPR / Cas9 system is combined with puromycin screening, the positive cloning rate reaches 30% or above, the construction period is shortened to 45 days, the constructed cell line can be directly used for AKAP11 related cell signal path research, disease model construction and targeted drug screening, and a reliable tool is provided for basic research and clinical transformation.

The invention discloses an endoglucanase mutant as well as a coding gene and application thereof. A mutant gene is obtained by carrying out semi-rational design on a wild endoglucanase A4 gene by adopting alanine scanning mutagenesis and saturation mutagenesis, and a recombinant expression vector is constructed. After the engineering bacterium escherichia coli Rosetta (DE3) is subjected to fermentation culture, mutant protein can be efficiently expressed. A high-efficiency expression system is established through genetic engineering, a new technology is provided for cellulose degradation and biomass resource utilization, and the method has application value in the field of biological energy sources. The results provide theoretical insights for enzyme activity and stability enhancement driven by mutation, and guide future enzyme engineering work.

This disclosure provides protein mutants generated using the large language program EVOLVE-Pro, which is designed for a wide range of applications. Examples of protein mutants include miniature CRISPR nucleases. EVOLVE-Pro substantially enhances the efficiency and effectiveness of in silico protein evolution, surpassing current state-of-the-art methods and yielding proteins with more than 100-fold improvement of desired properties. EVOLVE-Pro demonstrates the necessity for protein engineering models to zoom in on desired functional properties rather than predicted fitness, paving the way for broader applications of AI-guided protein engineering in biology and medicine.

The invention discloses a T7RNA polymerase mutant with improved specific enzyme activity and 3 '-consistency and application of the T7RNA polymerase mutant, and belongs to the field of gene engineering and enzyme engineering. Starting from a wild T7RNAP protein structure, a mutant with improved specific enzyme activity and 3 '-consistency is obtained. Compared with the prior art, the T7RNAP mutant provided by the invention has higher specific enzyme activity and thermal stability, generates lower dsRNA by-products, can tolerate higher reaction temperature, and has wide application prospects in the aspects of in-vitro transcription, circular RNA, mRNA vaccines, isothermal amplification, acellular in-vitro transcription and translation systems, gene editing and drug production research.

The application discloses a kind of construction methods of mouse model for studying vascular endothelial senescence, belong to genetic engineering technical field, utilize 6030442E23Rik conditional gene knockout mouse and Tek-CreERT2 mouse (utilize CRISPR gene editing technology, Cre is inserted into mouse Tek gene stop codon, is the tool mouse of endothelial cell conditional knockout, currently, this kind of strain mouse has on market), cross, obtain 6030442E23Rik- / -Tek-CreERT2 mouse.Tamoxifen is dissolved in corn oil later, so that its final concentration is 20 mg / ml.Tamoxifen dose is 120mg / kg mouse weight later, intraperitoneal injection 5 times, once every other day, and the mouse model of endothelial cell 6030442E23Rik specificity knockout can be obtained 7 days after the end of last injection.The mouse model of endothelial cell 6030442E23Rik specificity knockout is an important mouse model for studying vascular endothelial senescence.

The present disclosure provides compositions and methods of use of nucleic acids and complexes thereof targeted to nucleic acids. Genomic engineering can refer to the alteration of a genome by deletion, insertion, mutation, or replacement of a particular nucleic acid sequence. The alteration can be gene or position specific. Genomic engineering can utilize nucleases to cleave nucleic acids, thereby generating a site for alteration. Engineering of non-genomic nucleic acids is also contemplated.

The invention discloses a gene associated with cotton fiber length and application, and belongs to the field of biotechnology application. The cDNA (complementary deoxyribonucleic acid) sequence of the gene GhDTX48 associated with the cotton fiber length character is shown as SEQ ID NO. 1, and the genome sequence is shown as SEQ ID NO. 2. The gene GhDTX48 is regulated and controlled by an adjacent SNP site (D11: 24631774), the SNP basic group is mutated from T to C, and the length of genotype fiber after mutation is obviously greater than that before mutation. The invention further provides a method for identifying the variety of the long-fiber cotton, and the cotton with the basic group C corresponding to the SNP site is the variety of the long-fiber cotton. Meanwhile, a new variety of cotton long fibers is cultivated through a genetic engineering means, a gene containing a high-yield genotype GhDTX48 (C) is transferred into a cotton variety, or an SNP site in a low-yield genotype GhDTX48 (T) is subjected to site-specific mutagenesis to form (C), so that the new variety of cotton long fibers is cultivated. The gene has important research value and application prospect in efficient identification of long-fiber cotton varieties and improvement and breeding of excellent cotton varieties.

The present application relates to the technical field of genetic engineering, and particularly relates to a screening method of an anti-aging drug. The screening method comprises: treating cells overexpressing GFP-progerin protein with a drug to be screened, and comparing the cells overexpressing GFP-progerin protein without treatment of the drug to be screened; and judging the anti-aging effect of the drug to be screened according to the change of cell morphology. The present application finds that progerin protein positioned on the nuclear membrane can make cells show obvious aging characteristics, including nuclear membrane shrinkage and nuclear membrane budding. Progerin protein positioned in the nucleus does not make cells show obvious aging characteristics. When a drug has an anti-aging effect, progerin protein positioned on the nuclear membrane will be transferred to the nucleus. The present application provides a screening method of an anti-aging drug based on the above, which can accurately and efficiently screen an anti-aging drug.

The invention discloses a method for accurately editing key enzyme genes in a plant secondary metabolic pathway, belongs to the technical field of plant genetic engineering and metabolic engineering, and realizes accurate editing of a plurality of key enzyme genes in the plant secondary metabolic pathway by adopting a CRISPR / Cas9 technology through a multi-target synchronous editing strategy in combination with a tRNA-sgRNA tandem expression system and a homologous recombination repair mechanism. Through the optimized agrobacterium tumefaciens-mediated transformation method, the transformation efficiency is improved to 15-35%. The editing integrity is improved to 55-75%, and the off-target frequency is reduced to 0.3-1.2%. The method is applied to various plants such as sweet wormwood, tomatoes and catharanthus roseus, the content of secondary metabolites such as terpenoids, flavonoids and alkaloids is increased by 3-10 times, and a powerful technical support is provided for efficient production of the secondary metabolites of the plants.

Methods, polynucleotides, and compositions for generating engineered Treg cells are provided. The methods, polynucleotides and compositions enable reprogramming of hematopoietic cells into Treg cells by constitutive or regulatory expression of FOXP3 in engineered cells, such that the engineered Treg cells can inhibit activation and proliferation of responsive T cells.

Engineered CRISPR from Prevotella and Francisella 1 (Cpf1) nucleases with altered and improved target specificity and their use in genomic engineering, epigenomic engineering, genome targeting, genome editing, and in vitro diagnostics.

The application discloses an endoglucanase mutant EG06, a coding gene thereof, a product of the endoglucanase mutant EG06 and application thereof, and belongs to the field of genetic engineering and enzyme engineering. The technical problem to be solved is that the endoglucanase activity is low, and the heat resistance and acid resistance are poor in the prior art. The endoglucanase mutant EG06 has an amino acid sequence as shown in SEQ ID NO. 2. The sequence of EG03 as shown in SEQ ID NO. 1 is subjected to S153M, G189W, N193P and S209W mutations to obtain the endoglucanase mutant EG06. The endoglucanase mutant EG06 has outstanding performance at high temperature and in acidic pH, has the optimal pH 6.0, the optimal temperature 40 DEG C, and the high-efficiency catalytic characteristics of maintaining 30% enzyme activity at 20 DEG C, and has significant application potential in the industrial field.

The application discloses a method for synthesizing natural products by using methanol yeast chassis cells and an engineering strain, relates to the technical field of genetic engineering, and comprises a squalene engineering strain.The construction of the squalene engineering strain comprises the following steps: cloning a rate-limiting enzyme gene in Pichia pastoris GS115 into a vector pPICZA with a PAOX1 promoter, electrically transforming a linearized recombinant plasmid into the Pichia pastoris GS115, integrating the linearized recombinant plasmid into a genomic HIS4 site, screening positive transformants, and obtaining a first strain.The first strain is synthesized, the squalene synthesis capacity is established in the chassis cells, the problem that the wild-type Pichia pastoris has a very low endogenous mevalonate pathway flow is solved, the rate-limiting bottleneck of the squalene synthesis pathway is eliminated, and the precursor supply level is improved.

Systems and methods for highly-effective CRISPR-Cas based genomic editing within human chromosome 15q11-q13 have been developed as therapeutic interventions for Angelman Syndrome (AS). Selective single guide RNA molecules (sgRNAs) that impart enhanced CRISPR-Cas editing of genomic mutations associated with Angelman Syndrome in human cells are described. The engineered sgRNAs induce activity of non-pathogenic, paternal UBE3A alleles to reduce, reverse and / or prevent the causative neurodevelopmental defects of AS in a subject in need thereof. Compositions and methods of engineered crRNAs, sgRNAs thereof and ribonucleoprotein (RNP) complexes thereof are provided for enhanced genomic engineering with increased on-off target specificity and on-target editing efficacy for treatment of AS.

Engineered CRISPR from Prevotella and Francisella 1 (Cpf1) nucleases with altered and improved target specificity and their use in genomic engineering, epigenomic engineering, genome targeting, genome editing, and in vitro diagnostics.

The invention provides a CD26-targeted double-AAV delivery system based on a CRISPR base editing technology, and belongs to the field of gene engineering. According to the invention, a targeted CD26 editing system is constructed for the first time, in-vivo delivery is carried out through double AAV vectors, and selective removal of CD26 + CAFs in PDAC is successfully realized. The CD26 gene splicing donor site is precisely edited, the CD26 expression in the CAFs is remarkably reduced, lymphatic vessel generation and immunosuppressive cell infiltration are further reduced, LNM generation is blocked, and the tumor immune microenvironment is improved.

The invention provides a T-DNA complex as well as a preparation method and application thereof, and belongs to the technical field of genetic engineering. The invention provides a preparation method of a T-DNA (T-deoxyribonucleic acid) complex, which comprises the following steps: binding a single-chain T-DNA containing a target gene with RecA protein and VirD2 protein in sequence to obtain VirD2-RecA-ssT-DNA. The preparation method is simple in step and convenient to operate. The invention further provides a transgenic method of the T-DNA complex based on the scheme, the T-DNA complex VirD2-RecA-ssT-DNA is transferred into a plant receptor under the action of the cell permeable membrane peptide, a transgenic plant is obtained through screening, and the average transformation efficiency is about 6.1%. According to the transgenic method disclosed by the invention, a technical system for transforming the T-DNA complex into the gramineous plant is established, and a technical support is provided for functional genomics research of the gramineous plant.

The application belongs to the technical field of genetic engineering and cell modification, and discloses a genetic engineering construction method and application of an anti-apoptosis human mesenchymal stem cell. The application realizes a breakthrough improvement through specific lactic acidification mutation at the HDAC1 K412 site, for example, precise gene editing mediated by CRISPR-Cas9, only a single amino acid site is changed, that is, K412R mutation simulates continuous lactic acidification, and the whole genome epigenetic modification disturbance can be avoided; and the modification is not dependent on exogenous metabolic substrate, and the negative feedback effect of traditional metabolic intervention is overcome; the mutant continuously inhibits the P53 apoptosis pathway, and the ability of MSC to resist oxidative stress-mediated apoptosis is significantly improved. The design concept of "one target point and one regulation" fundamentally solves the core contradiction that the prior art cannot consider long-acting and specificity at the same time, and provides a new paradigm for stem cell therapy.

The invention discloses a method for constructing an escherichia coli whole genome CRISPRi library and an application of the escherichia coli whole genome CRISPRi library in target product synthesis, and belongs to the technical field of gene engineering. According to the invention, an sgRNA plasmid library is designed according to escherichia coli BL21 (DE3) genome information, and a sensor plasmid and an sgRNA library plasmid are simultaneously introduced into a tetR-Ptet-dCas9 expression strain to obtain a CRISPRi library which can be used for screening strains with high yield of target products. According to the present invention, by using lactyl-N-neotetraose as a model product, eight inhibition genes, such as cysW, pyrG, sprE, pcnB, YqjA, TnaB, TolB and ihfB, beneficial to the production of the lactyl-N-neotetraose are obtained; according to the invention, a screening method with feasibility is established, a key gene beneficial to synthesis of a target product is identified and obtained, and an efficient method is provided for enhancing synthesis of the target product of a strain.

The invention belongs to the technical field of genetic engineering and enzymology, and particularly relates to isopentenyl transferase and application thereof to preparation of isopentenyl isoflavone. The isopentenyl transferase FoPT2 with the amino acid sequence shown as SEQ ID NO.2 is verified for the first time to show catalytic activity on 6 isoflavone compounds (genistein, dihydrogenistein, biochanin A, calycosin, genistin and 3 ', 4', 7-trihydroxy isoflavone), 21 isopentenyl products are generated in total, the structures of 10 products are analyzed, and 3 products are synthesized for the first time. The isopentenylation modification comprises 13 kinds of mono-isopentenylation (C- / O-connection) and 8 kinds of diisopentenylation (C-O connection / C-C connection), modification sites comprise C-6, 7-O, C-8, 4 '-O and the like, and wide regioselectivity is shown. By optimizing reaction conditions, the catalytic efficiency of FoPT2 is improved, the preparation of isopentenyl isoflavone with various structures is facilitated, and a new possibility is provided for isopentenyl isoflavone products.

The invention discloses an endoglucanase mutant EG06 as well as a coding gene, a product and application thereof, and belongs to the field of gene engineering and enzyme engineering. The technical problems that in the prior art, endoglucanase is low in activity, poor in heat resistance and poor in acid resistance are solved. The endoglucanase mutant EG06 disclosed by the invention has an amino acid sequence as shown in SEQ ID NO.2, and the endoglucanase mutant EG06 is obtained by carrying out S153M, G189W, N193P and S209W mutation on an EG03 sequence as shown in SEQ ID NO.1. The invention further discloses a preparation method of the endoglucanase mutant EG06. The endoglucanase mutant EG06 shows prominent at high temperature and acidic pH, has the optimal pH of 6.0 and the optimal temperature of 40 DEG C, has the efficient catalytic characteristics of keeping 30% enzyme activity at 20 DEG C, and has remarkable application potential in the industrial field.

The invention discloses an NLRP3 gene knockout cell line as well as a construction method and application thereof, and belongs to the technical field of gene engineering. The NLRP3 gene is a gene for coding NLRP3 protein, and the NLRP3 protein is a constituent part of NLRP3 inflammasome and plays an important role in the process that cells respond to external stimulation and generate pyroptosis. According to the shRNA of the specific target NLRP3 gene provided by the invention, a J774A.1 cell line of which the NLRP3 gene is stably knocked out can be constructed. The invention discloses application of an NLRP3 gene knockout cell line, illustrates the key function of the NLRP3 gene in the processes of resisting pathogenic bacterium infection, responding to pyroptosis reaction induced by lipopolysaccharide LPS and adenosine triphosphate ATP and the like of a J774A.1 cell, and can also provide experience for other cell lines to obtain stably knocked-out cell lines.

Provided are methods and compositions for obtaining genome-engineered iPSCs, and derivative cells with stable and functional genome editing at selected sites. Also provided are cell populations or clonal cell lines derived from genome-engineered iPSCs, which comprise targeted integration of one or more exogenous polynucleotides, and / or in / dels in one or more selected endogenous genes.