59results about How to "Improve gene editing efficiency" patented technology

The invention employs a CRISPR-Cas9 system to complete mediation of a goat Tbeta4 gene for site-directed knock-in. A gRNA expression vector and a Tbeta 4 homologous recombinant vector based on the CRISPR-Cas9 system are constructed according to a CCR5 gene sequence of the goat. An optimized CRISPR-Cas9 vector and a constructed gRNA expression vector and the Tbeta 4 homologous recombinant vector are transferred to skeletal muscle satellite cells together, in order to obtain cells with the site-directed Tbeta4 gene. A targeting vector constructed based on the CRISPR-Cas9 system provides a simple, fast and safe pathway for site-directed knock-in of the goat Tbeta4 gene. Any screening marker genes are not related in a cell line screening process, safety of transgenic animals is greatly improved, and the method has important value for genetic breeding of goats and research of gene function.

The invention relates to an experimental method for constructing a Hutat2:Fc gene knock-in monocyte by using a CRISPR / Cas9 technology. The experimental method is characterized by comprising the following steps of (1) CRISPR / Cas9 targeting plasmid construction; (2) donor plasmid construction; (3) primary generation monocyte sorting; (4) monocyte transfection through electroporation; and (5) knock-in efficiency identification. The experimental method for constructing the Hutat2:Fc gene knock-in monocyte by using the CRISPR / Cas9 technology has the advantages that the method is simple and implemented easily, subsequent cell screening is not needed, the higher gene editing efficiency can be obtained, the feasibility and application prospect of the experiment are greatly increased, and the application range is wide.

The invention provides a novel enhancin CREnhancer 1.0 capable of increasing in vivo CRISPR / Cas9 gene editing efficiency obviously, provides a novel approach used for increasing gene editing efficiency in vivo, and provides a genome editing system with higher efficiency. When the enhancing and CRISPR / Cas9 are used together, CRISPR / Cas9 genome editing efficiency can be increased obviously.

The invention provides some new enhancins ESCS-higher, which can obviously improve the gene editing efficiency of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas9 in an epidermal stem cell. The invention provides a new way to improve the gene editing efficiency in the cell, and further provides a more efficient genome editing system. When the enhancin is used with the CRISPR / Cas9, the genome editing efficiency of the CRISPR / Cas9 in the epidermal stem cell can be obviously improved.

The invention discloses a method for knocking out a non-model organism plutella xylostella APN1 gene with CRISPR / Cas9 and an application of the method in molecular research on Bt Cry1Ac insecticidal protein resistance mechanisms. According to the method, a specific sgRNA target sequence of the plutella xylostella APN1 gene is designed and synthesized, sgRNA and Cas9 protein are mixed, the mixtureis microscopically injected into the posterior pole part of early embryonic disc stage eggs of plutella xylostella, plutella xylostella APN1 gene homozygous mutant populations with stable heredity arescreened out by a CRISPR / Cas9 gene editing system and applied to toxicity bioassay of Bt Cry1Ac insecticidal protein, which proves that plutella xylostella APN1 gene mutation can cause high resistance to Bt Cry1Ac insecticidal protein. The method is simple and efficient, time and labor are saved, technical support is provided for deep research on functional genes in non-model organisms, and a foundation is laid for exploring new pest control strategies.

The invention discloses a double sgRNA-mediated gene accurate modification method and an application thereof. According to the invention, two strips of sgRNA are simultaneously used, a CF cell is taken as a model, directional deletion is carried out on nucleotide of the 508th sites amino acid for coding CFTR gene, the pathogenic genotype which is same with the deletion patients of 508th sites amino acid of the CFTR can be obtained, and the method found that compared with single sgRNA, the double sgRNA has higher efficiency for editing. The invention provides the more effective method for cell model construction and gene editing for the gene disease.

The invention provides novel methods and compositions for enhancing gene editing.

The invention discloses a method for constructing high-quality porcine nuclear transplantation donor cells with high lean meat percentage, fast growth and resistant to porcine reproductive and respiratory syndrome and series diarrhea diseases and application of the high-quality porcine nuclear transplantation donor cells. A CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR associated protein 9) system for porcine MSTN-SST-CD163-pAPN four-gene editing contains a Cas9 expression vector and gRNA (guide Ribonucleic Acid) expression vectors aiming at a porcine MSTN gene, an SST gene, a CD163 gene and a pAPN gene, and plasmid complete sequence of the Cas9 expression vector is as shown in SEQ ID NO.2. According to the invention, the corresponding gRNA expression vectors are designed for different targets of MSTN, SST, CD163 and pAPN genes, and gRNA with relatively high editing efficiency and the gRNA expression vector are obtained through screening. Through cooperation with the modified Cas9 efficient expression vector for gene editing, the editing efficiency is remarkably improved as compared with that of an original vector.

The invention discloses a method for verifying the feasibility of inserting a CRISPR-Cas9 system mediated target gene into candida utilis. The method includes the following steps that S1, an expression vector of the candida utilis containing Cas9 segments is constructed; S2, tRNAGlu genes and downstream expression units of sgRNA are amplified, PCR link is overlapped and a restriction enzyme cutting site is introduced, and after connection, the amplified tRNAGlu genes and downstream expression units of the sgRNA are combined with the vector of the candida utilis to obtain sgRNA initial recombinant plasmid; S3, pre-insertion and target sequences are selected, a sticky tail end is added to the 5' end of a target and complementary sequence, and double chain segments are formed by annealing andthen connected with the initial recombinant plasmid after enzyme digestion to obtain sgRNA mature recombinant plasmid; S4, a recombinant donor segment containing a left homologous arm and a right homologous arm of a pre-insertion sequence is constructed; and S5, the sgRNA recombinant plasmid and the recombinant donor segment are used for transforming the candida utilis carrying Cas9. The feasibility of inserting the CRISPR-Cas9 system mediated target gene into the candida utilis is verified by identifying the expression of the donor segment.

The invention relates to the technical field of biology and particularly provides a composition for fixed-point modification of a pAPN-gene 16 exon and application of the composition. The composition for the fixed-point modification of the pAPN-gene 16 exon, provided by the invention, comprises a targeted enzyme digestion vector 1, a targeted enzyme digestion vector 2 and a double-stranded donor sequence. A modified pAPN gene can effectively resist TGEV infection, meanwhile, the protein physiological activity function of the modified pAPN gene is not affected, and the modified pAPN gene has the advantages of being wide in application range, high in gene editing efficiency and the like.

The invention discloses a method for constructing high-quality porcine nuclear transplantation donor cells with high lean meat percentage, fast growth, high fertility and resistance to series epidemic diseases and application thereof. A CRISPR / Cas9 system for editing five genes of porcine MSTN, SST, INHA, CD163 and pAPN comprises a Cas9 expression vector and gRNA expression vectors respectively aiming at the porcine MSTN, SST, INHA, CD163 and pAPN genes; and the plasmid complete sequence of the Cas9 expression vector is shown as SEQ ID NO. 2. The corresponding gRNA expression vectors are respectively designed aiming at different target points of the MSTN, SST, INHA, CD163 and pAPN genes, and gRNA with higher editing efficiency and expression vectors thereof are obtained through screening. The gene editing is performed by matching with the modified Cas9 high-efficiency expression vector, and the editing efficiency is obviously improved compared with that of the original vector.

The invention provides a gene editing microneedle for treating inflammatory skin diseases and application thereof. The gene editing microneedle comprises a needle tip part and a base part, wherein the needle tip part comprises two nano compounds and a needle tip matrix, and the base part is a base matrix. The gene editing microneedle can improve the gene editing efficiency, deliver the retention of glucocorticoid drugs and gene editing ribonucleoprotein compounds in the skin, and synergistically improve the skin inflammation environment. The microneedle has the skin barrier function of repairing skin lesion tissues, thereby achieving the inflammatory process of relieving psoriasis and atopic dermatitis. The invention also provides the application of the gene editing microneedle in the preparation of drugs for treating inflammatory skin diseases.

The invention provides a method for detecting salmonella. The method comprises the following steps: (1) taking nucleic acid of a sample to be detected as a template, and carrying out amplification bya loop-mediated isothermal amplification technique by using specific primers of the salmonella, so as to obtain an amplified product; (2) under mediation of specific crRNA, subjecting the amplified product obtained in the step (1) to a cracking reaction by using a CRISPR system, so as to obtain a cracked product; and (3) carrying out chromogenic detection on the cracked product obtained in the step (2) by using a colloidal gold test paper strip. The method has very good sensitivity and accuracy.

The present invention provides synthetic single guide RNAs that comprise two separate functional sequences (commonly known as crRNA and tracrRNA) connected by a linker. These synthetic single guide RNA molecules are useful in gene editing when used with RNA-guided endonucleases such as cas9 in eukaryotic cells. The availability of the synthetic single guide RNAs makes the screening for gene editing in high-through-put format simple and convenient.

The present invention provides synthetic single guide RNAs that comprise two separate functional sequences (commonly known as crRNA and tracrRNA) connected by a linker. These synthetic single guide RNA molecules are useful in gene editing when used with RNA-guided endonucleases such as cas9 in eukaryotic cells. The availability of the synthetic single guide RNAs makes the screening for gene editing in high-through-put format simple and convenient.

The invention discloses a method for knocking out an NANS gene in iPSC (induced pluripotent stem cells) and application, and belongs to the field of cell model construction. According to the invention, a CRIPSR-Cas9 gene editing system is transferred into the iPSC by adopting an electroporation method; Donor DNA containing a Puromycin resistance gene and a GFP gene is inserted into an NANS exon, and the stem cells subjected to gene editing are screened by Puromycin; and after transfection, CloneRTM culture cells are added, and the system can greatly improve gene editing efficiency of a CRISPR-Cas9 genome editing technology in the stem cells. The method is helpful for constructing an NANS defect type 3D brain organ model, and can be applied to in-vitro study and drug research and development on the basis of pathogenesis of mental development disorders caused by the NANS mutation.

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 relates to the field of gene editing, in particular to wheat gene editing, and discloses a method for optimizing wheat editing efficiency. By the method, wheat gene editing efficiency isincreased.

The invention relates to the technical field of gene editing, and particularly relates to application of a chemical modification CRISPR / Cpf1 compound. The invention discloses application of the CRISPR / Cpf1 compound in cell gene editing. In the compound, chemical modification Cpf1 protein is coupled with crRNA and is marked as cCpf1. The compound is used for gene editing, so that the gene editing efficiency can be improved. When the compound is applied to the preparation of immune cells, the preparation efficiency of CAR-T can be improved; more importantly, the preparation efficiency of multi-site gene editing and universal CAR-T can be improved.

The invention discloses application of a CRISPR / Cas9 genome editing system in alfalfa gene editing, the CRISPR / Cas9 genome editing system comprises an expression vector, the expression vector can comprise a Cas9 expression cassette, an sgRNA1 expression cassette and an sgRNA2 expression cassette, and the Cas9 expression cassette is used for expressing Cas9. The sgRNA1 expression cassette is used for expressing sgRNA with the name of sgRNA1, and the sgRNA2 expression cassette is used for expressing sgRNA with the name of sgRNA2; the sgRNA1 expression cassette contains a promoter with the name of MtU6-6promoter and a sgRNA1 gene driven by the MtU6-6promoter, and the sgRNA2 expression cassette contains a promoter with the name of MtU6-5promoter and a sgRNA2 gene driven by the MtU6-5promoter; the sgRNA1 and the sgRNA2 can aim at the same target gene or different target genes of the alfalfa. According to the invention, double targets are designed for a target gene according to the characteristics of an autotetraploid of alfalfa, a gene editing binary vector p6401-Target is constructed, and alfalfa is subjected to agrobacterium-mediated transformation to obtain a regenerated plant. According to the optimized genome editing system, the alfalfa gene editing efficiency is greatly improved, the plant gene editing efficiency can reach up to 100%, and the single target editing efficiency can reach up to 96.9%.

The invention relates to a method for improving activity of a gene knockout and base editing system by using a small molecule compound Rociinostat or Nexturastat A. When a gene editing system is used for gene editing, the small molecule compound Rociinostat or Nexturastat A is added to treat cells, and the Rociinostat or Nexturastat A improves activity of the gene knockout and base editing system in the gene editing system. The method of the invention obtains higher gene editing efficiency, solves a problem of low activity of specific gene editing at precise sites, and is helpful for clinical tumor treatment target screening, drug development, disease-causing mutation correction, animal model preparation, and the like.

The invention provides a composition, application, cell and gene-editing pig preparation method for modifying an amino acid 561 of a CD163 gene, and relates to the technical field of biology. The composition for modifying the amino acid 561 of the CD163 gene comprises recombinant plasmids and single-stranded donor sequences. The recombinant plasmids comprise gene-editing vector skeletons and a section of DNA sequences connected to the vector skeletons. The single-stranded donor sequences are single-stranded DNA sequences in which the amino acid 561 of the CD163 gene is precisely replaced, theamino acid 561of the CD163 gene is precisely modified, and meanwhile, the situation that the normal expression of remaining amino acids of CD163 gene is destroyed or changed can be avoided; and therefore, on the basis of resisting PRRSV infection, the physiological activity function of CD163 protein is retained to the greatest extent, and the advantages of wide application range and high gene-editing efficiency are achieved.

The invention belongs to the technical field of gene engineering, and particularly relates to a method for preparing a citrus homozygous mutant by a CRISPR / Cas9 mediated gene editing technology. The method comprises the following steps of: co-culturing a citrus epicotyl stem and agrobacterium, immediately performing heat treatment on the epicotyl stem, and then transferring the epicotyl stem into an MS culture medium added with 1mg / L BA, 0.5mg / L IAA and 40mg / L Km for induction. The method can improve the gene editing efficiency of the homozygous mutant.

The invention discloses an OPG gene editing system for constructing an osteoporosis cloned porcine nuclear donor cell line and an application of the OPG gene editing system. The invention discloses a CRISPR / Cas9 system for pig OPG gene editing. The CRISPR / Cas9 system comprises a Cas9 expression vector and a gRNA expression vector, the Cas9 expression vector is a pKG-GE3 vector of which the plasmid complete sequence is as shown in SEQ ID NO. 2; the gRNA expression vector is used for expressing gRNA of which the sequence is shown as SEQ ID NO: 49. When the screened gRNA combined modified Cas9 efficient expression vector is used for gene editing, the editing efficiency is remarkably improved compared with that of the original vector.

The invention provides an MIC3 gene knockout recombinant coccidiosis vector and a detection method thereof, and belongs to the technical field of gene engineering. The invention provides sgRNA of a targeted coccidium MIC3 gene. The target spot of the sgRNA of the targeted coccidium MIC3 gene is located at the MIC3 gene; a gene editing system is established by sgRNA, Cas9 and homologous recombinant plasmids, coccidiosis MIC3 genes can be accurately knocked out, fluorescence-labeled protein can be homologous recombined to the MIC3 genes, and a recombinant coccidiosis vector for expressing the fluorescence-labeled protein is constructed. The DNA of the recombinant coccidiosis vector is subjected to PCR (Polymerase Chain Reaction) detection and sequencing to indicate that the fluorescence-labeled gene is successfully recombined with the MIC3 gene, and the recombinant coccidiosis vector has an obvious fluorescence signal under a fluorescence microscope to indicate that the MIC3 gene is successfully knocked out and the fluorescence-labeled protein is expressed.

A CRISPR system is successfully used to modify the genomes of a gram-positive bacterium, such as a species of the Corynebacterium genus. Methods for modifying Corynebacterium species include single-nucleotide changes, creating gene deletions and / or insertions.

Provided are an engineered CRISPR / Cas12f1 system of which editing efficiency is improved compared to a canonical CRISPR / Cas12f1 system, and a use thereof. A U-rich tail sequence in crRNA is provided that can be introduced into an original guide RNA to improve the editing efficiency of the CRISPR / Cas12f1 system. The U-rich tail sequence has abundant uridine residues at the 3′-ternimus of crRNA, and may further include additional nucleic acids in addition to uridine depending on the environment in which the guide RNA is actually used and the environment in which the guide RNA is expressed. The inventors of the present application described the structure of the U-rich tail sequence, the position of introduction, and the effect thereof with respect to genome editing efficiency.