109 results about "Frameshift mutation" patented technology

The invention discloses a method of increasing the content of resistant starch in rice through genome editing and sgRNA special for the same. The method provides a method of increasing the content of resistant starch and/or amylase in rice seeds. The method includes the following steps that expression of SBEIIb genes in the rice is inhibited; the SBEIIb genes are genes of encoding SBEIIb protein. The invention further provides a method of increasing the content of resistant starch in rice and includes the step of inhibiting the activity of the SBEIIb protein in the rice. By using the CRISPR/Cas9 technology, the rice SBEIIb genes are edited at fixed points, the rice SBEIIb genes are knocked off by causing frameshift mutation, and the new generation of new rice germplasm with the content of amylase and resistant starch obviously increased is obtained.

The present invention discloses a method for reducing rice amylose content through gene editing and a special sgRNA, and belongs to the field of biotechnology. The method comprises the following steps: expression of a Waxy gene in rice is inhibited; and the Waxy gene is the gene encoding Waxy protein. A CRISPR/Cas9 technology is utilized to edit the rice Waxy gene on site. By causing frameshift mutations and knocking out the rice Waxy gene, a new generation of rice germplasm with significantly reduced amylose content is obtained. Compared with a wild type, the obtained Waxy site-edited strainis significantly lowered in amylose content.

The invention discloses a sgRNA fragment. The DNA sequence of the sgRNA fragment comprises a length of a fixed sequence and a length of a DNA recognition sequence. The sgRNA can recognize different target sites on double strands of a target gene, bond with nuclease and guide nuclease to bond with the target sites of the target gene through recognition of PAM sequences at the target sites and to start random shearing; then nuclease falls off from a shearing opening, thereby forming a DSB gap; then cells repair the double strands of the target gene through a non-homologous end joining repair mechanism, which leads to frameshift mutation; and finally, the target gene is knocked out. The invention further discloses a gene fixed-point reconstruction method and a gene targeting kit. The invention elaborates a series of sgRNAs applicable to the mouse Myod1 gene, and the sgRNAs have high targeting efficiency, short construction time and low cost and can be highly efficiently used for mouse Myod1 gene targeting.

The invention discloses a method for introducing frame-shift mutation in bovine MSTN (myostatin) genes. According to the method, a targeting vector for the frame-shift mutation of the bovine MSTN genes is established, and the frame-shift mutation is introduced into the third exon of the bovine MSTN genes by virtue of homologous recombination. The method comprises the following steps of: synthesizing a homologous short arm containing the frame-shift mutation of MSTN, and allowing the homologous short arm to be short of 11bp on the third exon to form the frame-shift mutation; synthesizing a homologous long arm of the MSTN, and inserting a vector pFPC-1 by virtue of a restriction enzyme cutting site to establish the targeting vector pFPC-MSTN of the frame-shift mutation in the bovine MSTN genes; and transfecting bovine somatic cells after the linearization of the targeting vector, and introducing the frame-shift mutation in the bovine MSTN genes by virtue of the homologous recombination. Transgenic cattle with the frame-shift mutation of the MSTN genes can be obtained in the production by virtue of the cells, thereby improving beef production traits of the cattle and improving the quality and yield of beef.

The invention discloses a construction method of an HDAC8 gene knockout BHK-21 cell line, which comprises the following steps: carrying out gene knockout on HDAC8 in a cell line BHK-21 for foot-and-mouth disease vaccine production by utilizing a CRISPR/Cas9 technology, transfecting BHK-21 cells by utilizing CRISPR plasmids for knocking out the HDAC8, and performing separation to obtain a plurality of cell clones by utilizing antibiotic screening in combination with gradient dilution and a cloning ring method. After genome DNA is extracted, PCR amplification and sequencing are carried out, and cell clones of which two HDAC8 genes are subjected to homozygous frameshift mutation are successfully identified. In the HDAC8 knockout BHK-21 cell line, the replication rate of foot-and-mouth disease virus is obviously accelerated, the final virus titer is obviously improved, and the HDAC8 knockout has no obvious influence on the cell growth rate, which shows that the HDAC8 knockout BHK-21 cell line has the prospect of being used for foot-and-mouth disease vaccine production. A foundation is laid for further knocking out HDAC8 from suspension culture type BHK-21 cells and directly applying HDAC8 to production of foot-and-mouth disease vaccines.

The invention discloses a construction method of an HDAC3 gene knockout BHK-21 cell line, which comprises the following steps: carrying out gene knockout on HDAC3 in a cell line BHK-21 for foot-and-mouth disease vaccine production by utilizing a CRISPR/Cas9 technology, transfecting BHK-21 cells by utilizing CRISPR plasmids for knocking out the HDAC3, and separating the cells to obtain a plurality of cell clones by utilizing antibiotic screening in combination with gradient dilution and a cloning ring method; extracting the genome DNA, performing PCR amplification and sequencing, and successfully identifying the cell clones of which two HDAC3 genes are subjected to homozygous frameshift mutation. In the HDAC3 knockout BHK-21 cell line, the replication rate of foot-and-mouth disease virus is obviously accelerated, the final virus titer is obviously improved, and the HDAC3 knockout has no obvious influence on the cell growth rate, so that the HDAC3 knockout BHK-21 cell line has the prospect of being used for foot-and-mouth disease vaccine production. Therefore, the foundation is laid for further knocking out HDAC3 from suspension culture type BHK-21 cells and directly applying HDAC3 to foot-and-mouth disease vaccine production.

The invention discloses an HCBP 6 (Hepatitis C Virus Core-Binding Protein 6) gene knockout cell line and a construction method thereof. The construction method for the HCBP 6 gene knockout cell line is characterized in that a CRISPER/Cas9 (CRISPR associated protein 9) technology is adopted to construct the HCBP 6 gene knockout cell line, a fragment which conforms to a 5'-N[X]-NGG-3' or 5'-CNN-N[X]-3' sequence arrangement rule in the coding sequence of HCPB6 protein is taken as a target sequence, wherein N presents any one of A, G, C and T, X is greater than or equal to 14 or less than or equalto 30, in addition, X is an integer, and N[X] presents X continuous deoxyribonucleotide. The construction method lays a foundation for HCV (hepatitis C virus) infection mechanism research. The HCBP 6gene knockout constructed by the invention forms frameshift mutation on a genome level, the gene can be transferred to the next generation along with the division and the proliferation of cells, anda stable HCBP 6 gene knockout cell line is formed.

The invention belongs to the field of gene engineering and medical oncology and discloses BRCA1 gene g.41244291delT mutation and its application in breast cancer auxiliary diagnosis. The mutation BRCA1 gene sequence has a g.41244291delT site frame-shift mutation in comparison with BRCA1 normal gene sequence. The invention provides a new breast cancer-causing mutation site, and the mutation site can be used in early diagnosis of breast cancer.

The invention discloses a construction method of an HDAC5 gene knockout BHK-21 cell line, which comprises the following steps: carrying out gene knockout on HDAC5 in a cell line BHK-21 for foot-and-mouth disease vaccine production by utilizing a CRISPR/Cas9 technology, transfecting BHK-21 cells by utilizing CRISPR plasmids for knocking out the HDAC5, and separating the cells to obtain a plurality of cell clones by utilizing antibiotic screening in combination with gradient dilution and a cloning ring method; extracting the genome DNA, performing PCR amplification and sequencing and successfully identifying the cell cloning of which one HDAC5 gene is subjected to homozygous frameshift mutation, wherein the HDAC5-KO-A2 has deletion of 13 basic groups at a Cas9 predetermined cutting position. In the HDAC5 knockout BHK-21 cell line, the replication rate of foot-and-mouth disease virus is obviously accelerated, the final virus titer is obviously improved, and the HDAC5 knockout has no obvious influence on the cell growth rate, so that the HDAC5 knockout BHK-21 cell line has the prospect of being used for foot-and-mouth disease vaccine production. Therefore, the foundation is laid for further knocking out HDAC5 from suspension culture type BHK-21 cells and directly applying HDAC5 to production of foot-and-mouth disease vaccines.

The invention discloses a zinc finger nuclease-mediated porcine MSTN (myostatin) gene mutation sequence and application thereof. By mediating the porcine MSTN gene by the zinc finger nuclease technique, base deletion is enabled, frameshift mutation is resulted in, early termination of translation is caused, an MSTN functional protein is unable to be formed, and the MSTN gene mutation sequence with 3782th to 3796th nucleotides deleted and 3800th nucleotide molecules mutated from T to G is acquired. Experiments show that a pig with the MSTN gene having the 3782th to 3796th nucleotides deleted and the 3800th nucleotide molecules mutated from T to G has evident double-muscled phenotype, both muscle content and lean percentage are evidently increased, and fat content is evidently lowered.

The invention relates to a method for cultivating a new variety of normal-development intermuscular-bone-free fish, and relates to a method for cultivating a new variety of intermuscular-bone-free fish. The invention provides the method for removing intermuscular bones under the condition that normal growth of fishes is not influenced. The method for cultivating the new variety of the normal-development intermuscular-bone-free fish comprises the following steps of 1, knocking out a bmp6 gene in a fertilized egg of an original fish variety to obtain an F0-generation individual; 2, performing mutant screening on the F0 generation, and matching to obtain an F1 generation population; 3, performing mutant screening on the F1 generation population, selecting individuals with the same frameshift mutation for mating, constructing an F2 generation, and screening out homozygous mutation individuals in the F2 generation; and 4, propagating the F2-generation homozygous mutation individuals to obtain the new variety of the intermuscular-bone-free fish. According to the method, a strain with intermuscular bones deleted can be obtained, growth is not affected, stable inheritance can be achieved, and the problems that the intermuscular bones are small, difficult to find and difficult to remove in production can be solved.

The invention discloses a method for creating a red rice strain by targeting a Rc gene by using a gene edition technique. The method comprises the following steps: editing sequences of a rc gene frameshift mutation point by using a gene edition technique, screening and collecting an RC genotype with function recovery, and successfully improving rice colors of two materials, namely nonglutinous rice Gangyou 8518 and japonica rice Xiushui 134. By adopting the method, an efficient breeding mode is provided for creating red rice germplasm resources and culturing excellent red rice varieties.

The invention discloses a construction method of an HDAC2 gene knockout BHK-21 cell line. The construction method comprises the following steps: carrying out gene knockout on HDAC2 in a cell line BHK-21 for foot-and-mouth disease vaccine production by utilizing a CRISPR/Cas9 technology; the inventor transfects BHK-21 cells with CRISPR plasmids for knocking out HDAC2, and then separates a plurality of cell clones by using a cloning ring method by combining antibiotic screening with gradient dilution; after genome DNA is extracted, PCR amplification and sequencing are carried out, and cell cloning of which one HDAC2 gene is subjected to homozygous frameshift mutation is successfully recognized; wherein the HDAC2-KO-B3 has the deletion of one basic group at the predetermined cutting position of the Cas9. In the BHK-21 cell line with HDAC2 knockout, the replication rate of foot-and-mouth disease virus is obviously accelerated, the final virus titer is obviously improved, and the cell growth rate is not obviously influenced after HDAC2 knockout, so that the HDAC2 knockout BHK-21 cell line has the prospect of being used for foot-and-mouth disease vaccine production. A foundation is laid for further knocking out HDAC2 from suspension culture type BHK-21 cells and directly applying HDAC2 to production of foot-and-mouth disease vaccines.

The invention discloses a method for detecting the cutting efficiency of a gene editing target. The method comprises the following steps of constructing a target detection vector containing a frame shift mutation reporter gene; inserting a gene editing target sequence into an N end of the target detection vector containing the frame shift mutation reporter gene to obtain a detection vector; and transferring the detection vector into animal cells, repairing the mutation reporter gene by the cells when endonuclease successfully cuts the target, and detecting the activity of the reporter gene toobtain the target cutting efficiency. The method is sensitive and visual in detection and can be quantified; and target cutting occurs in the cells, which is extremely similar to actual gene editing reaction conditions, and can reflect the target cutting effect in actual gene editing.

The invention belongs to the field of gene engineering and relates to a pre-T vector and a T vector composed of the pre-T vector and application thereof. The improved promoter is a recognition site for mutation from a nucleotide sequence from -35 region to -10 region in a promoter region into endonuclease. By the T vector, the problem that the product of transcription or translation of exogenous genes by a strong promoter during blue-white selection of a vector might be toxic to host and cloning will fail can be overcome, blue-white selection of a vector might be toxic to host as a strong promoter starts transcription or translation; the defect that delection of 1-2 bp at enzyme digestion site leads to lacZalpha gene frameshift mutation and false positive clones are generated can be avoided; and the false negative phenomenon that a plate is full of blue stains because exogenous DNA fragment is small and insertion of exogenous DNA does not change a reading frame of lacZalpha gene can beeliminated.

The present invention provides a filter carrier for screening an oligonucleotide having a target length, the filter carrier, from 5 'to 3' direction, includes a promoter, a reporter gene, and at least one enzyme digestion site located between the promoter and the reporter gene, and after the oligonucleotide having the target length is inserted into the enzyme digestion site, frameshift mutation of the reporter gene may not be caused. The present invention also provides a construction method of the filter carrier and application of the filter carrier in improvement of correct rate of chemical synthesis oligonucleotides used in a phage or bacterial gene library construction process.