34results about How to "Achieve knockout" patented technology

The invention relates to a rapid construction method of a conditional gene knockout animal model, which is implemented by using a CRISPR / Cas9 technology. The specific method can be implemented as follows: designing a gRNA target spot sequence, for a to-be-knocked-out gene of an animal, of a CRISPR / Cas9 system, designing a corresponding promoter, constructing a plasmid, thereby implementing conditional gene knockout. According to the invention, the rapid, efficient, simple, feasible, economic, efficient, and wide-applicable-scope construction of a conditional gene knockout animal model can be realized, and the defects existing in traditional methods (specific recombination enzyme system Cre-LoxP) can be overcome.

The invention relates to a sgRNA targeting sequence and application, in particular to a specifically-targeted swine IGFBP3 gene sgRNA targeting sequence and application. The sgRNA targeting sequence is CGTCTCGCGCTTGGACTCAG. By the sgRNA targeting sequence, an IGFBP3 gene can be knocked out or edited through a CTISPR / Cas9 system so as to eliminate IGFBP3 expression to lay the foundation for preparation of IGFBP3 transgenic swine. The specifically-targeted swine IGFBP3 gene sgRNA targeting sequence is applied to the field of gene engineering.

The invention relates to an artificial gene editing system for the gene editing of rice. The artificial gene editing system comprises a regulation element I and a regulation element II, wherein the regulation element I comprises a nucleotide sequence which can encode an amino acid sequence I; the amino acid sequence I comprises one of amino acid sequence I-1, amino acid sequence I-2 and amino acidsequence I-3; the regulation element II comprises a nucleotide sequence II-1 and a nucleotide sequence II-2 in series connection in turn from the end 5' to the end 3'; the nucleotide sequence II-1 comprises a target nucleotide sequence; the target nucleotide sequence is derived from the genome of a target organism and contains the to-be-mutated target site in the genome of the target organism; the nucleotide sequence II-2 comprises sgRNA nucleotide sequence derived from streptococcus pyogenes; the nucleotide sequence II-1 and the nucleotide sequence II-2 are subjected to transcriptional fusion.

The invention discloses a primer for gene knockout, including five sets of primers, such as SEQ NO:1 and SEQ NO:2, SEQ NO:3 and SEQ NO:4, SEQ NO:5 and SEQ NO:6 , SEQ NO:7 and SEQ NO:8, SEQ NO:9 and SEQ NO:10. The invention also discloses the application of the primer in gene knockout. The invention also discloses a conditional gene knockout method based on CRISPR / Cas9 technology. The invention can carry out condition specificity, space-time specificity, and drug-induced gene modification; reduce the harm to other cells, and study the function of constitutively expressed genes in specific tissues; only the Cas9 tool mouse can realize tissue and space specificity Sexual gene knockout or inducible gene knockout; the test cycle is short, saving time and cost.

Fungus has wide applications in biotechnology. CRISPR / Cas9 system is a powerful genome editing method and is successfully applied to a plurality of genomes, but the system is not widely used in fungus, and thus, a convenient, effective and widely applicable method which uses CRISPR / Cas9 system to perform gene editing in fungus is provided.

The invention provides a sgRNA combination of a targeted AHRR gene and application of the sgRNA combination, the sgRNA combination of the targeted AHRR gene comprises sgRNA1 and / or sgRNA2, the sgRNA1 comprises a nucleic acid sequence as shown in SEQ ID No.1, and the sgRNA2 comprises a nucleic acid sequence as shown in SEQ ID No.2. The invention also provides an AHRR gene editing system, a recombinant cell and a construction method thereof, and a construction method of an obese animal model, through gene editing and screening, the obtained homozygous mouse without the AHRR gene has typical obesity signs, larger body type, heavier body weight, higher fat content in vivo, and can be used for screening blood sugar and lipid reducing related drugs, and has extremely wide application value.

The invention discloses an sgRNA directing sequence for specific targeting of a human ABCG2 gene and an application thereof, and belongs to the field of gene engineering application. The sgRNA directing sequence has a nucleotide sequence of GCTGCAAGGAAAGATCCAAG. According to the sgRNA directing sequence, two single-stranded oligo sequences are designed and synthesized and then are annealed to form a double chain, and then the double chain is connected with a Cas9 vector; with use of the Cas9 vector, sgRNA and a CRISPR system are introduced into target cells, a Cas9 protein can find out a DNA sequence matched with the Cas9 protein under guidance of the sgRNA, shearing is performed, and the ABCG2 gene is knocked out. According to the sgRNA directing sequence provided by the invention, the ABCG2 gene is knocked out or edited by the CRISPR-Cas9 system, then the expression of the ABCG2 is inhibited or eliminated, and the problem of multi-drug resistance generated in treatment of tumor can be effectively solved.

The invention provides a method for enhancing the genome editing efficiency of Streptomyces and its application. By adding elements that control Cas9 activity, the triple control of Cas9 protein activity at the transcription, translation, and protein levels can be achieved, the cytotoxicity of Cas9 protein to Streptomyces can be inhibited, and the transformation efficiency of plasmids in Streptomyces can be improved. Achieve efficient gene editing. The present invention establishes a gene editing system that is both induced by small chemical molecules and regulated by inhibitory proteins, which can inhibit the toxicity of Cas9 in vivo, and realize the efficient introduction of genetic elements without affecting the transformation efficiency of edited plasmids and the growth and metabolism of host cells. and subsequent gene editing. Small-molecule chemical inducers are easy to add, and can realize efficient and precise genome editing based on double-strand breaks and directional repair. The present invention can be applied to Streptomyces genetic engineering and genetic modification including model or industrial Streptomyces.

The invention provides an establishing method of a recoverable immortalized hepatic cell line. The establishing method comprises the following steps: separating a hepatic progenitor cell from liver in a mouse which is 12.5-14.5 days old in an embryonic period; guiding a retrovirus containing genes SV40T and HSV-TK into the hepatic progenitor cell; screening a monoclonal cell strain having a hepatic progenitor cell marker and having a function of differing to a mature hepatic cell; then guiding a retrovirus containing CD gene into the cell strain so as to obtain recoverable immortalized hepatic cell carrying double suicide genes. The cell strain can multiply in vitro to obtain the phenotype and functions of a normal hepatic cell, the safety of the immortalized cell becomes adjustable through controllable modes such as locus recombination and drug screening; when the cell is used in a human body, the biological safety of the immortalized cell is ensured to the greatest extent and the dangerousness of the immortalized cell is reduced; therefore, a reliable, safe and ideal hepatic cell material is provided for bioartificial liver technology.

The invention discloses a method for increasing the ovalbumin expression of Bacillus subtilis, and belongs to the field of genetic engineering. The invention improves the ovalbumin expression of Bacillus subtilis by increasing the specific growth rate of Bacillus subtilis. In the present invention, the wild-type 168 is used as the starting strain, and a plurality of recombinant strains with increased specific growth rate can be obtained by using the method of knockout of growth-related genes, adaptive evolution (Adaptive Laboratory Evolution, ALE) and the combination of the two. The specific growth rate of Bacillus to increase the expression of Bacillus subtilis ovalbumin provides a new idea and method for increasing the expression of Bacillus subtilis ovalbumin.

The invention discloses three newly isolated fast-growing Vibrio strains and applications thereof, and relates to the field of bioengineering. The three Vibrio strains are named Vibrio sp.FA1, Vibrio sp.FA2 and Vibrio sp.FA3 respectively, and were published on August 2019. It was deposited on the 5th in the China Center for Type Culture Collection, address: Wuhan University, China, and the deposit numbers are CCTCC NO: M 2019603, CCTCC NO: M 2019604, CCTCC NO: M 2019605. Compared with the known fastest growing Vibrio natriegens ATCC 14048, these three Vibrio strains have obvious growth advantages. Through genome-wide comparative analysis, it is found that they also have more DNA replication-related genes, amino acid synthesis-related genes and resistance Inversion-related genes; these three Vibrio strains can grow rapidly using a variety of carbon sources under the condition of chemically synthesized medium, which is more conducive to industrial application. In addition, the transformation plasmids and gene knockout by electrotransformation proved the genetic operability of these strains, which provided an important basis and basis for the application of these three strains of Vibrio in the field of industrial technology.

The present invention provides a combination of sgRNA targeting AHRR gene and application thereof, the combination of sgRNA targeting AHRR gene includes sgRNA1 and / or sgRNA2, and the sgRNA1 includes the nucleic acid sequence shown in SEQ ID No. 1, and the sgRNA2 includes the nucleic acid sequence shown in SEQ ID No.2. The present invention also provides an AHRR gene editing system, a recombinant cell and a method for constructing the same, and a method for constructing an obese animal model. Obesity signs, larger body size, heavier body weight, and higher body fat content can be used in the screening of hypoglycemic and lipid-lowering related drugs, and have extremely wide application value.

The present application relates to a set of artificial gene editing system for gene editing of rice, comprising: a regulatory element I comprising a nucleotide sequence capable of encoding an amino acid sequence I, the amino acid sequence I comprising one of an amino acid sequence I-1, an amino acid sequence I-2 and an amino acid sequence I-3; and a regulatory element II which comprises an II-1 nucleotide sequence and an II-2 nucleotide sequence which are sequentially connected in series from the 5' end to the 3' end. The II-1 nucleotide sequence comprises a target nucleotide sequence; the target nucleotide sequence is derived from a genome of a target organism, and the target nucleotide sequence contains a target site to be mutated in the genome of the target organism; the (II-2)th nucleotide sequence comprises an sgRNA nucleic acid sequence derived from streptococcus pyogenes; and the (II-1)th nucleotide sequence and the (II-2)th nucleotide sequence are transcribed and fused.

The invention belongs to the field of biotechnology and provides a systemic Plin1 gene knockout animal model construction and identification method thereof. The steps of the construction method are: designing sgRNA sequences according to the sequence before and after exon 2 of the Plin1 gene, constructing a Plin1 gene knockout vector, and performing in vitro transcription to obtain sgRNA; mixing sgRNA and Cas9 protein and then microinjecting it into mouse fertilized eggs to obtain whole body Sexual Plin1 knockout mice. The beneficial effect of this project is that a pair of sgRNA sequences with the highest scores were screened out, which ensured the success rate of the project; a large fragment of exon 2 of the Plin1 gene was knocked out, and the knockout efficiency was improved; the knockout region was designed 3 The two primers can successfully identify various genotypes of mice, avoiding the situation that two primers may not be able to distinguish genotypes when large fragments are knocked out. It provides a convenient, reliable and economical animal model and a good foundation for studying the biological function of PLIN1 and the pathogenesis of obesity-related metabolic diseases.