13results about How to "Improve gene editing efficiency" patented technology

The invention discloses a hansenula polymorpha efficient gene editing system based on CRISPR (clustered regularly interspaced short palindromic repeats) / Cpf1 and application of the hansenula polymorpha efficient gene editing system. The hansenula polymorpha gene editing system comprises: (1) a Cpf1 integration plasmid, wherein the Cpf1 integration plasmid is expressed by an endogenous constitutive promoter, and the Cpf1 is integrated into a hansenula polymorpha genome; (2) a crRNA expression plasmid, wherein the crRNA expression plasmid comprises a crRNA expression cassette; wherein the crRNA expression cassette is composed of an RNA pol III type promoter and a terminator.

The present invention provides an engineered CRISPR enzyme and system for precise gene editing and its application, wherein the CRISPR enzyme has an amino acid sequence as shown in SEQ ID No: 1, and the gene editing system comprises a CRISPR fusion protein and a guide RNA component. The present invention is used for gene editing, gene targeting, gene cutting, preparation of gene-edited animals, establishment of animal disease models, establishment of disease tissues, preservation and transformation of animal genetic species resources of eukaryotic cells, achieving efficient gene editing, with predictable editing products and very few by-products, thus making up for the shortcomings of existing gene editing tools.

The invention relates to an RNA (Ribonucleic Acid) coding element and a method for carrying out gene editing on a plant by using the RNA coding element. In particular to an RNA (Ribonucleic Acid) coding element, an sgRNA bidirectional enhanced expression element, a carrier capable of carrying out efficient gene editing in a plant and a method for carrying out gene editing on the plant. The vector can improve the gene editing efficiency and the targeting specificity of the plant, and the sgRNA bidirectional enhanced expression sequence can further improve the gene editing efficiency and the targeting specificity of the plant.

This invention discloses the application of chloroquine in improving gene editing efficiency. Specifically, it provides a novel use for chloroquine: enhancing the gene editing efficiency of gene editing reagents on recipient biological materials. The gene editing reagent is a gene editing tool plasmid. No prior art studies have combined chloroquine with gene editing efficiency. The inventors of this invention have discovered that treating recipient biological materials with chloroquine before transfecting them with gene editing reagents can significantly improve gene editing efficiency. This invention is simple to operate, widely applicable, and opens up a new avenue for improving editing efficiency by introducing exogenous small molecules rather than directly modifying gene editing tools.

The invention discloses a construction method and application of a novel coronavirus animal infection model, and relates to the fields of molecular biology technology and animal gene engineering, the virus replicon prepared by the invention can complete multiple rounds of replication only in specific cells stably expressing novel coronavirus SARS-CoV-2N protein, but cannot be replicated in other cells, and the virus replicon can be used as a novel coronavirus animal infection model. The biological safety risk is greatly reduced, and the system can be operated in a BSL-2 laboratory. Besides, the invention successfully constructs a safe novel coronavirus SARS-CoV-2 animal infection model capable of being used in a BSL-2 laboratory by utilizing an SARS-CoV-2 trans complementary replication subsystem without an N gene and a mouse model for expressing SARS-CoV-2N protein in infected tissue specificity or specific time. The method can be applied to screening, research and development of novel coronavirus resisting drugs.

The invention provides a novel gene editing protein Cas12a-X, a mutant Cas12a-X1 of the novel gene editing protein Cas12a-X and application of the mutant Cas12a-X1. According to the invention, a new gene editing protein Cas12a-X is explored from a metagenome of human intestinal microorganisms, and the homology of the gene editing protein Cas12a-X and common LbCas12a is 48%. Through the guidance of crRNA, the novel gene editor shows remarkable in-vitro and in-vivo editing activity, the editing efficiency is equivalent to that of LbCas12a, and the application value is high. Based on the structure and functional characteristics of a wild type Cas12a-X protein, mutation is carried out on amino acid sites of a key structural domain of the wild type Cas12a-X protein to obtain a Cas12a-X1 three-protruding variant. In-vivo experiments prove that the gene editing efficiency of the mutant is improved by at least 1.2 times compared with that of wild type Cas12a-X. The application range of the CRISPR-Cas12a system is expanded, and a new tool is provided for the fields of precision medicine, disease treatment, variety improvement and the like.

The invention discloses a multi-PAM-selection efficient gene editing system NovaCas-1 and application thereof, and belongs to the technical field of gene editing. The gene editing system NovaCas-1 is a complex of NovaCas-1 protein and sgRNA, and can accurately locate a targeted DNA sequence and generate cleavage to enable DNA to generate double-strand breakage damage, the amino acid sequence of the NovaCas-1 protein is as shown in SEQ ID NO: 1, and the nucleotide sequence of the sgRNA is as shown in SEQ ID NO: 7 or SEQ ID NO: 9. The NovaCas-1 protein has the beneficial effects that (1) as many as 47 PAM sequences can be recognized by the NovaCas-1 protein, so that the target gene editing range of the existing gene editing system is greatly widened; and (2) experiments prove that the gene editing system NovaCas-1 has higher gene editing efficiency than the existing gene editing system.

The invention discloses a recombinant plasmid based on a nuclear localization signal and a PP7 RNA (Ribonucleic Acid) stem-loop structure, the recombinant plasmid comprises NLS-cas9-NLS + 6 * PP7 RNA, and the gene sequence of the NLS-cas9-NLS + 6 * PP7 RNA is as shown in SEQ ID NO. 2. The invention also discloses an application of the recombinant plasmid in VLP, exosome, lentivirus or AAV delivery vectors. The invention also discloses an efficient VLP carrier system, which comprises the recombinant plasmid, and also comprises a plasmid for fusion expression of PP7 bacteriophage capsid protein and gag structural protein, a plasmid for expression of membrane protein, an auxiliary plasmid, a shuttle plasmid carrying gRNA and an integrase mutated pMDLg-pRRE plasmid. The invention also discloses application of the vector system in treatment of clinical diseases. The vector system can improve the gene editing efficiency.

The invention belongs to the technical field of biology, and discloses a small molecule compound and a method for improving homologous recombination efficiency of pichia pastoris aiming at the problem of low homologous recombination efficiency of pichia pastoris, and the small molecule compound comprises at least one of compounds SCR7, EPZ5676 and nocodazole. The method for improving homologous recombination efficiency of pichia pastoris provided by the invention comprises the following steps: mixing a constructed OPI1 gene targeting recombinant vector with a purified OPI1 knockout donor DNA fragment, adding the mixture into prepared pichia pastoris competent cells, carrying out electric shock transformation, adding a small molecule compound, and carrying out standing recovery and culture to obtain the homologous recombination efficiency of the pichia pastoris. The recombinant pichia pastoris strain is obtained. According to the invention, small molecule compounds SCR7, EPZ5676 and nocodazole are applied to a pichia pastoris CRISPR / Cas9 system, and the homologous recombination efficiency of pichia pastoris is obviously improved by regulating and controlling a repair way and a cell cycle.

The invention discloses a multi-PAM selection efficient gene editing system NovaCas-3 and application thereof, and belongs to the technical field of gene editing. The gene editing system NovaCas-3 is a complex of NovaCas-3 protein and sgRNA, and can accurately locate a targeted DNA sequence and generate cleavage to enable DNA to generate double-strand breakage damage, the amino acid sequence of the NovaCas-3 protein is as shown in SEQ ID NO: 1, and the nucleotide sequence of the sgRNA is as shown in SEQ ID NO: 7 or SEQ ID NO: 9. The NovaCas-3 protein has the beneficial effects that (1) as many as 38 PAM sequences can be recognized by the NovaCas-3 protein, so that the target gene editing range of the existing gene editing system is greatly widened; and (2) experiments prove that the gene editing system NovaCas-3 has higher gene editing efficiency than the existing gene editing system.

The invention relates to the field of immunotherapy, in particular to a nucleic acid construct containing a nucleotide sequence of a synthetic T cell antigen receptor (STAR) encoding a specific binding tumor-associated antigen, and an engineering T cell prepared from the nucleic acid construct. The invention also relates to application of the nucleic acid construct and the engineering T cell in tumor treatment.

The present application belongs to the technical field of crop breeding, and relates to a potato breeding method for editing ALS gene. The method comprises the following steps: (1) using agrobacterium transformed with a gene editing tool to infect potato explants, wherein the infection medium used contains CaCl2 with a total concentration of 9-15 mM, and the gene editing tool targets potato ALS gene for editing mutation of ALS gene to make potato resistant to imidazolinone herbicides; (2) after co-cultivation and recovery culture, the potato explants are sequentially subjected to resistant callus induction, resistant bud induction and seedling culture on resistant callus induction medium, resistant bud induction medium and seedling medium to obtain regenerated seedlings, and the resistant callus induction medium, resistant bud induction medium and seedling medium all contain imidazolinone herbicides. By using the method of the present application, potato varieties with ALS gene mutation can be obtained with high infection efficiency and high gene editing efficiency.

The invention discloses a multi-PAM selection efficient gene editing system NovaCas-2 and application thereof, and belongs to the technical field of gene editing. The gene editing system NovaCas-2 is a complex of NovaCas-2 protein and sgRNA, and can accurately locate a targeted DNA sequence and generate cleavage to enable DNA to generate double-strand breakage damage, the amino acid sequence of the NovaCas-2 protein is as shown in SEQ ID NO: 1, and the nucleotide sequence of the sgRNA is as shown in SEQ ID NO: 7 or SEQ ID NO: 9. The NovaCas-2 protein has the beneficial effects that (1) as many as 36 PAM sequences can be recognized by the NovaCas-2 protein, so that the target gene editing range of the existing gene editing system is greatly widened; and (2) experiments prove that the gene editing system NovaCas-2 has higher gene editing efficiency than the existing gene editing system.