35results about How to "High mutation efficiency" patented technology

The invention provides a mutant protein based on activated induced cytidine deaminase. The mutant protein is obtained by mutating hsAID as follows: T82I, K10E, K34E, E156G, 181 *, S38, H130, V152, R174 and T100. The invention also provides a single base site-specific editing protein. The protein comprises the mutant protein based on activated induced cytidine deaminase and a DNA specific binding protein, wherein the mutant protein based on activated induced cytidine deaminase and the DNA specific binding protein are sequentially connected through a connecting sequence. The invention also provides a single base site-specific editing system. The system comprises the single base site-specific editing protein and a targeted high mutation sequence. Compared with the existing single base editingsystem based on activated induced cytidine deaminase (AID), the induced mutant protein system provided by the invention has the advantages of smaller molecular weight and higher mutation efficiency.

The invention discloses a method for constructing gene site-directed mutation. The method comprises the following steps: determining a target sequence which is in a rat target genome sequence and can be identified by an artificial modified CRISPR-Cas (clustered regularly interspaced short palindromic repeats-associated protein) system; constructing a gRNA (guide ribonucleic acid) which can identify a CAS protein and guide the CAS protein to a target gene target sequence; introducing the gRNA sequence and a CAS protein coded deoxyribonucleic acid sequence or the gRNA sequence and an in-vitro expressed CAS protein mixture into a rat embryonic cell. According to the method, a homologous recombined targeting vector does not need to be constructed, ES (embryonic stem) targeting cell screening is not needed, a mutant has a high proportion, the operation is convenient, simultaneous multi-site knockout can be realized, the experiment cost can be greatly reduced, and the experiment cycle can be shortened.

The invention provides a high-efficiency preparation method for simultaneously preparing multiple member mutants of the same gene family of cabbage mustard. The preparation method comprises the following steps: according to the whole length of each member of the gene family of the cabbage mustard, analyzing a target site of each member, and screening a common target sequence; constructing a CRISPR / Cas9 expression vector according to the selected target sequence, then transforming into agrobacterium competent state, infecting explants, and then performing tissue culture on the explants to obtain mutant plants. Multiple genes of the same gene family in a cabbage mustard plant are simultaneously knocked out through once transformation, and a large number of mutant materials can be obtained through mutation of different members, so that the mutation efficiency is improved, the workload is saved, the working time is saved, the problems that in past gene family mutation, multiple vectors arerequired to be designed respectively and the vectors are required to be separately transformed so as to obtain different mono-mutant and multi-mutant materials, induction is long in time and high indifficulty and the like are solved, and thus the advantages of simultaneousness and high efficiency of the preparation method are embodied.

The invention discloses a method for editing rice giant embryo gene GE to cultivate giant embryo japonica rice varieties. The present invention uses the CRISPR-Cas9 multi-target system to control the size of rice embryos GE The megaembryo gene is edited at a fixed point, and the japonica rice genetic transformation is carried out to obtain the megaembryo transgenic plant, and the detection of the transgene insertion fragment in the offspring can shorten the breeding cycle and quickly obtain a variety of japonica rice varieties with large and small embryos without the insertion of the transgene fragment. same embryo size the ge The GABA content of the brown rice of the genotype mutants is also different, and the GABA content also increases with the growth of the embryo. Rice varieties with different embryo weights can satisfy the extraction of different GABA contents to meet the needs of different consumer groups. Therefore, the present invention can provide an efficient gene knockout method and a method for cultivating multiple giant embryo japonica rice varieties for production applications with different GABA content requirements.

The invention discloses a method for site-directed mutation of alfalfa gene by using CRISPR / Cas9 system. The present invention uses CRISPR / Cas9 technology in the cross-pollination autotetraploid plant alfalfa for the first time, and obtains mutant plants; for the first time, the MtU6 promoter is used to initiate sgRNA transcription in alfalfa to improve transcription efficiency; and natural mutation and traditional Compared with artificially induced mutations, the present invention accurately introduces mutations with higher mutation efficiency, and can achieve a mutation rate of 52%, of which the homozygous mutation rate reaches 12%. The invention directly mutates the gene coding sequence, which opens up a new field for gene editing in alfalfa, and lays the foundation for subsequent simultaneous silencing of multiple genes, deletion of large chromosome fragments, precise insertion of foreign genes into target sites, gene regulation, etc. technical basis.

The present invention provides a CHO cell that can be used for protein display and expression. An expression cassette is integrated into the genome of the CHO cell, and the expression cassette is integrated after the stop codon of the YWHAE gene in the CHO cell genome; the expression cassette Including promoter, first recombinase recognition sequence, target gene, second recombinase recognition sequence, terminator. In this cell, the fluorescent protein gene and antibody gene can be highly transcribed and continuously and stably expressed in the absence of antibiotics, and the antibody transcription level has been significantly improved compared to the previously established CHO-puro cell line. It was verified by experiments that the AID mutation efficiency of this site was significantly higher than that of the random insertion site of CHO-puro.