70 results about "Genomic engineering" patented technology

Engineered CRISPR-Cas9 nucleases with altered and improved PAM specificities and their use in genomic engineering, epigenomic engineering, and genome targeting.

The invention discloses a glutamate decarboxylase multi-site combined mutant and application thereof, and relates to the technical field of enzyme engineering and gene engineering. Amino acid residue sites needing to be mutated are determined on the basis of amino acid sites near key amino acids of a coenzyme 5 '-phosphopyridoxal binding pocket, and experimental verification is performed through a site-directed mutagenesis technology in combination with a high performance liquid chromatography screening method. Compared with a chemical synthesis method, the compound is synthesized by utilizing glutamic acid decarboxylase through biological catalysis, the synthesis condition is mild, the operation is simple, and separation and extraction are easy. According to the method, the screening probability of mutant amino acid sites can be effectively improved, the experiment efficiency and feasibility are improved, and the mutant enzyme of which the enzyme activity is obviously superior to that of the parent enzyme is obtained through screening.

The invention belongs to the technical field of gene engineering and enzyme engineering, and particularly relates to a glucanase mutant and construction and application of a gene sequence of the glucanase mutant, and the glucanase mutant has an amino acid sequence as shown in SEQ ID NO.2. The glucanase mutant is obtained by carrying out G214A mutation, S249P mutation and D286L mutation on an amino acid sequence as shown in SEQ ID NO. 1. The glucanase mutant has the advantages that the glucanase mutant is obtained; compared with the non-mutated EGL, the glucanase mutant EGL62 provided by the invention has the advantages that the thermal stability is obviously improved; after incubation at 75 DEG C for 3 minutes, the enzyme activity of 60% or above can still be maintained; the enzyme activity is still more than 50% after the enzyme is treated for 1 hour under the condition that the pH value is 3.0-7.0. The glucanase mutant EGL62 provided by the invention has the optimal pH value of 5.0 and the optimal temperature of 50 DEG C, and is good in stability and wide in application prospect.

Disclosed herein are system-related compositions based on clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) 9 and methods of using the system-related compositions based on CRISPR / Cas9 for altering gene expression and genome engineering. Also disclosed herein are compositions and methods of using the compositions for altering gene expression and genome engineering in muscles such as skeletal muscle and cardiac muscle.

The invention discloses a method for improving synthesis of sakuranetin from yarrowia lipolytica based on transcriptome analysis and application of the method, and belongs to the technical field of gene engineering. Target genes are analyzed and screened through transcriptome sequencing so as to find a method capable of improving sakuranetin synthesis, and the method specifically comprises the steps that an Illumina HiseqTM platform is used for conducting comprehensive transcriptome analysis on an original strain in the logarithmic phase by applying an RNA-seq technology, then transcriptome data are deeply analyzed through KEGG pathway analysis, and the sakuranetin synthesis can be improved. Therefore, the gene showing significant differential expression in the key path of sakuranetin synthesis is identified. And finally, carrying out free expression and integrated expression on the screened key target gene, and verifying the influence on the sakuranetin yield. Finally, a recombinant yarrowia lipolytica strain which is integrated with the YALI0C01859g gene and has high sakuranetin yield is obtained, and the titer of sakuranetin is increased to 344.0 mg / L.

The invention discloses an incision beta-1, 3-glucanase mutant with improved enzyme activity and thermal stability, and belongs to the technical field of gene engineering and enzyme engineering. According to the invention, a site-directed mutagenesis technology is utilized to carry out molecular modification on wild type beta-1, 3-glucanase, and a series of mutants S22F, N226D and N187S are obtained. Compared with a wild enzyme, the beta-1, 3-glucanase mutant constructed by the invention has the advantages that the enzyme activity is improved by 1.04-1.81 times, the Tm value is improved by 0.97-1.19 DEG C, and the half-life period at 70 DEG C is prolonged to 1.08-1.28 times. According to the invention, the enzyme activity and the thermal stability of the wild beta-1, 3-glucanase are improved at the same time, and better use conditions are created for the enzyme in practical application.

The invention relates to the field of gene engineering, in particular to a proximity marker enzyme mutant and application thereof. The proximity marker enzyme mutant takes ProtA-Turbo as a starting sequence, and one or more sites in K71, K240, K335, K345 and K351 are mutated into non-polar hydrophobic amino acid, polar neutral amino acid, acidic amino acid, basic amino acid or non-natural amino acid. The proximity marker enzyme mutant overcomes the influence of self biotinylation on the proximity marker activity, can continuously and stably mark the proximity proteome of the target protein on the immobilized cell, and obviously improves the sensitivity of identifying the proximity interacting protein of the target protein.

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 discloses a mutant of glutamate decarboxylase as well as a recombinant thallus and application thereof, and belongs to the field of protein engineering. According to the invention, a glutamate decarboxylase mutant M5 is obtained through a gene engineering method; the optimal temperature of the enzyme is increased by 10 DEG C while the thermal stability of the glutamate decarboxylase mutant is improved, and the mutant can play a very good catalytic effect without being in a buffer salt solution, so that an effective way is provided for industrial production of gamma-aminobutyric acid by an enzyme method; meanwhile, the problem that the product yield is low under the conventional production condition due to low thermal stability or too low optimal catalytic temperature when the glutamate decarboxylase is applied is also solved to a great extent.

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 invention discloses a process application method for improving the yield of butenyl spinosad, and belongs to the technical field of genetic engineering, the process application method comprises the following steps: taking a strain genome of a mutant strain S.pogona aG6 as a template, constructing to obtain an engineering strain O1322-6746, researching the influence of different factors on the yield of butenyl spinosad of the engineering strain O1322-6746, selecting the optimal factor, and selecting the optimal factor to improve the yield of butenyl spinosad. The method comprises the following steps: performing a single-factor experiment on components of the culture medium, optimizing the concentration of each component of the culture medium through the single-factor experiment, optimizing the yield of butenyl spinosad according to the result of the single-factor experiment to obtain a regression equation, and applying the optimized culture medium to amplified fermentation of an O1322-6746 strain in a 5L bioreactor. The accuracy of the equation model for predicting the yield of the butenyl spinosad is verified, and the yield of the butenyl spinosad of the engineering strain O1322-6746 can be improved.

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 present invention relates to engineered CRISPR-Cas9 nucleases. Engineered CRISPR-Cas9 nucleases with improved specificity and their use in genome engineering, epigenome engineering, genome targeting, and genome editing are disclosed.

The invention belongs to the technical field of gene engineering, and provides an MSC specific promoter LY6E and application thereof, the MSC specific promoter LY6E is selected from one of SEQ ID NO: 1 to SEQ ID NO: 9, preferably SEQ ID NO: 7, or a sequence having at least 95.8% similarity with the sequence of SEQ ID NO: 7, or a sequence having at least 86.4% similarity with a truncated fragment of SEQ ID NO: 7. The MSC specific promoter provided by the invention can carry an exogenous gene to enter cells in a pluripotent stem cell stage, only starts transcriptional activity after the pluripotent stem cells are differentiated into the MSC, and is of great significance to MSC engineering modification, research on MSC-based precise treatment and the like.

The invention discloses a plasmid vector for overexpressing a YES1 gene as well as a preparation method and application of the plasmid vector, and relates to the technical field of molecular biotechnology and genetic engineering. The invention provides a plasmid vector for overexpressing the YES1 gene, overexpression of the YES1 gene can be stably and effectively realized by transfecting the plasmid vector in a host cell, so that cell activity can be enhanced, and cell proliferation, migration, invasion, increase of oxygen consumption rate and transformation (EMT) from epithelial cells to mesenchymal cells can be promoted; therefore, when the plasmid vector of the overexpressed YES1 gene is applied to preparation of drugs for regulating and controlling cell metabolism and fibrosis, the plasmid vector has very high practical application value.

The present invention discloses the application of a β-galactosidase gene and the enzyme encoded thereby, belonging to the technical fields of genetic engineering and enzyme engineering. The present invention screened a β-galactosidase with a specific amino acid sequence and successfully carried out heterologous expression of the β-galactosidase in Escherichia coli. The method of the present invention is safe and efficient, and the catalytic activity of the crude enzyme solution of the expressed β-galactosidase can reach 12378.6 U / mg, which can be applied to the food and pharmaceutical fields. This β-galactosidase is used to convert lactose to produce galactooligosaccharides, wherein the conversion rate can reach 70.9%, and the proportion of galactooligosaccharides generated can reach 63.1%, which is beneficial to industrial production.

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 invention belongs to the field of gene engineering, and particularly discloses a recombinant expression plasmid vector for expressing TNFSF11 protein and a construction method and application of the recombinant expression plasmid vector, a nucleotide sequence contained in the plasmid vector is optimized according to host cell preferred codons under the condition that an amino acid coding sequence is not changed, and the nucleotide sequence is shown as SEQ ID NO: 1. Furthermore, an expression vector of the nucleotide sequence is constructed, and the recombinant TNFSF11 protein is expressed in an HEK293 cell; the concentration of the expressed recombinant TNFSF11 protein is 3.04 mg / mL, the activity is 2.47 * 10 < 6 > U / mg, and the purity is 99%. The expression quantity and biological activity of the soluble protein are improved, and conditions are created for large-scale production of the soluble recombinant TNFSF11 protein.

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 invention relates to the field of genetic engineering and protein modification technology, and proposes a high specific activity alpha-amylase mutant and its application. The present invention is based on wild-type alpha-amylase A1, and provides a mutant comprising at least one mutation site in N39T, P145N, D159Y, F191N, Q240S, D279N, R306S, T310N, H319T, and I477V. Compared with wild-type alpha-amylase A1, the specific activity of the xylanase mutant provided by the present invention is generally increased by 26.2%-35.7%; wherein, the specific activity of the alpha-amylase mutant containing the T310N single-point mutation is the highest, reaching 1251.38U / mg, which is conducive to reducing the production cost of the enzyme and promoting its wide application in the industrial field.

The present invention relates to a P450 enzyme capable of hydroxylating the C11α position of progesterone, its gene, expression vector, cell and application, belonging to the technical fields of enzyme engineering and genetic engineering. The progesterone P450 hydroxylase CYP BMH3 and its gene sequence cyp bmh3 derived from the prokaryotic microorganism Bacillus megaterium H-1. The full length of this gene is 1233 bp, encoding 410 amino acids, and its amino acid sequence is shown in SEQ ID NO.1, and the gene sequence is shown in SEQ ID NO.2; and based on the heterologous expression of this enzyme, the efficient conversion of progesterone is achieved, with high specificity, reduced reaction by-products, and reduced production costs, laying a foundation for its further industrial application.

The present invention relates to the field of genetic engineering technology, and provides an mRNA-fatty acid targeting compound, a preparation method thereof and an application. The present invention uses an oligo-adenylic acid-fatty acid compound to carry out an RNA ligation reaction with a target mRNA to obtain an mRNA-fatty acid targeting compound. The present invention provides a brand-new mRNA-fatty acid targeting compound, which solves the problem that fatty acids cannot be efficiently coupled with mRNA, and the coding target protein of the mRNA-fatty acid targeting compound has a high specific expression level in muscle tissue and can be directly delivered to muscle cells by intramuscular injection or systemic injection.

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 belongs to the technical field of gene engineering, and discloses an efficient miniature CRISPR / Cas9 gene editing system and application. The amino acid sequence of the Cas9 protein disclosed by the invention is as shown in SEQ ID NO. 1; or the amino acid sequence shown as SEQ ID NO.1 is a sequence obtained by substitution, deletion or insertion of at least one amino acid in the amino acid sequence shown as SEQ ID NO.1. According to the Cas9 protein disclosed by the invention, the protein structure is simplified to the greatest extent while the core catalytic activity is maintained, and the editing efficiency of the Cas9 protein in different application scenes can be improved; the method can effectively target a genome region where a non-standard PAM sequence is located, and the editing ability of a CRISPR system in a complex genome is improved. A new thought is provided for diversified development of a gene editing technology.