5results about How to "Improve gene editing efficiency" patented technology

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 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 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 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.