44 results about "Frameshift mutation" patented technology

The invention discloses a rice salt stress resistant gene OsHAK11 coding protein and application thereof, and belongs to the field of plant genetic engineering. According to the method, a rice OsHAK11 gene (the nucleotide sequence is shown as SEQ ID NO.1) is knocked out through a CRISPR / Cas9 gene editing technology, and a mutant with significantly enhanced salt tolerance is obtained. The gene editing vector pEGCas9Pubi-B-OsHAK11 contains sgRNA of a region as shown in a target SEQ ID NO.4, a rice receptor material is transformed through agrobacterium tumefaciens mediation, and a plant with the OsHAK11 gene subjected to frame shift mutation is obtained through screening. A salt stress experiment shows that the survival rate of the mutant oshak11 is obviously higher than that of a wild type. The invention provides a new gene resource and an efficient technical means for salt-tolerant breeding of rice.

The invention discloses an oilseed rape gene BnaKNAT1s and an application thereof in creation of droop-type silique oilseed rape, and belongs to the technical field of plant genetic engineering. The gene comprises two copies of BnaA03.KNAT1 and BnaC03.KNAT1, and the CDS (Coding Sequence) sequences of the gene are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2. Targeted sgRNA is designed according to homologous regions of first exons of the two genes, nucleotide sequences of the sgRNA are shown as SEQ ID NO: 3 and SEQ ID NO: 4 respectively, CRISPR / Cas9 editing vectors are constructed, agrobacterium tumefaciens is transferred, and then rape is infected, so that a transgenic plant is obtained. And screening a homozygous mutant with two genes subjected to frameshift mutation and without a transgenic element, hybridizing the homozygous mutant with a good variety, and carrying out multi-round backcross and selfing to obtain a new germplasm of the sagittaria silique rape. According to the germplasm, the canopy structure is optimized, the light energy utilization rate and ventilation performance are improved, high-density planting is adapted, and gene resources and core materials are provided for high-yield breeding of oilseed rape.

The invention relates to the technical field of biology, and particularly discloses a method for regulating expression of an SH3.1 gene to reduce rice seed shattering and application of the method. The method for reducing the seed shattering of the rice comprises the following steps: inhibiting the expression of an SH3.1 gene in receptor rice to obtain target rice of which the seed shattering is lower than that of the receptor rice. The inventor adopts a CRISPR / Cas9 technology to edit an SH3.1 gene at a fixed point, and knocks out the SH3.1 gene of an introgression line GIL291 of an African cultivated rice IRGC104038 segment by causing frameshift mutation, so that a new rice germplasm with obviously reduced seed shattering is obtained. High seed shattering is an important factor restricting yield increase of African cultivated rice, the rice seed shattering is reduced, high-yield germplasm resources of rice are innovated, and the method has important significance on new variety cultivation, environmental sanitation and grain safety. The method has great application and popularization values.

The present invention provides a method for improving rice yield tms5 The method for the sterility threshold temperature of two-line sterile lines comprises the following steps: TMS5 CRISPR / Cas9 gene editing targets T501 and T502 were designed 22bp and 57bp downstream of the gene translation start site ATG, respectively, and gene editing vectors TMS501 and TMS502 were constructed to transform rice varieties to achieve TMS5 Site-directed mutation of genes to obtain different multiple alleles tms5 Gene frameshift mutant material. Experiments have shown that in the same genetic background, the T501 target site editing tms5-1 The sterility starting temperature of the frameshift mutants was significantly higher than that of the T502 target. tm5-2 Mutants. The method provided by the present invention can quickly obtain rice with different sterility threshold temperature phenotypes under the same genetic background. tms5 Mutants can overcome the interference of different genetic backgrounds and provide a basis for studying rice tms5 The molecular mechanism and genetic regulatory network of the starting temperature of two-line sterility provide new ideas.

The invention discloses a method for preparing a CD163 gene editing pig based on a CRISPR (clustered regularly interspaced short palindromic repeats) / Gs12-7MAX system. The method specifically comprises two editing strategies: one is to edit a sixth intron and a seventh intron of a CD163 gene to realize accurate deletion of a seventh exon so as to obtain a porcine fibroblast monoclonal cell with homozygous deletion of the seventh exon; and 2, editing aiming at a seventh exon of the CD163 gene, and inducing the exon to generate frame-shift mutation to obtain a gene editing pig. The technical system established by the invention has the advantages of high editing efficiency, strong accuracy and simplified operation process, provides a new effective strategy for prevention and control of PRRSV (porcine reproductive and respiratory syndrome), and has important popularization and application values in creation of new materials for pig disease-resistant breeding.

The present application relates to a kind of gRNA targeted to non-human animal OPG gene, preparation method of OPG gene knockout non-human animal model.The preparation method of OPG gene knockout non-human animal model includes the following steps: using gene editing technology, destroy non-human animal OPG gene, for the gRNA targeted to non-human animal OPG gene includes one or two shown in SEQ ID NO:1 And / or SEQ ID NO:2.The present application designs the gRNA specifically targeted to non-human animal OPG gene, using Cas9 protein, OPG gene is knocked out, causes frame shift mutation, to reach the purpose of gene knockout to non-human animal.The method of the present application to non-human animal OPG gene modification is simple and easy to operate, short cycle, using the method of OPG model for research OPG gene physiological mechanism and molecular mechanism provides foundation, while providing important tool for research osteoporosis pathogenesis and drug development.

The invention discloses a method for constructing an animal model of a nasal mucosa specific expression target protein. The invention provides a method for constructing a mouse model of a nasal mucosa specific expression target protein, which comprises the following steps: replacing an Obp1a gene termination codon in a mouse genome with a target protein coding gene without causing frameshift mutation, thereby obtaining the mouse model of the nasal mucosa specific expression target protein. By adopting an optimized gene editing technology and a nasal mucosa Obp1a specific promoter, the target protein is ensured to be highly expressed only in a nasal mucosa area, the specific gene expression of the nasal mucosa is accurately regulated and controlled, an efficient and specific animal model is constructed, the efficient, stable and specific expression of the target protein in nasal mucosa tissues is realized, and the application prospect is wide. Therefore, reliable experimental tools and technical supports are provided for follow-up nasal disease mechanism research, new drug screening and gene function analysis.

The invention provides a gene for regulating nitrogen absorption and root growth under salt stress and application of the gene. OsGSK1 gene is knocked out from rice to obtain OsGSK1 mutant rice; the nucleotide sequence of the OsGSK1 gene is as shown in SEQ ID NO: 1. According to the invention, the OsGSK1 gene in rice is knocked out to generate frame-shift mutation, and the OsGSK1 mutant rice is obtained. A mutant strain is compared with a wild strain, the lateral root density of the mutant in a high-salt environment is remarkably higher than Taizhong 65, and meanwhile, the total nitrogen content and the nitrogen absorption and utilization rate of the overground part of the mutant strain are also remarkably higher than Taizhong 65. The results show that the rice OsGSK1 gene has potential application value in improvement of nitrogen utilization under rice salt stress, and has important significance in stress-resistant breeding of rice and improvement of rice yield.

This invention discloses a method for rapidly creating high-quality fresh soybean germplasm using gene molecular marker-assisted screening, relating to the field of genetic engineering technology. Specific primers were designed targeting frameshift mutation editing sites in the GmLOX1, GmLOX2, GmLOX3, GmBadh1, and GmBadh2 genes. These specific primers amplify only the edited genotype and not the wild-type. This method can assist in screening for fresh soybeans that are free of beany odor and have a pleasant aroma, and can also distinguish between edited and wild-type soybeans. The method is simple to operate, highly efficient, and has a short screening cycle.

This invention belongs to the field of rice molecular breeding, specifically relating to a method for cultivating rice with high resistant starch content; it constructs... SS3a and SS3b Gene co-knockout vectors were created and screened using Agrobacterium-mediated methods to obtain simultaneous knockouts. SS3a and SS3b A new rice germplasm containing the gene; sequencing results of PCR amplification products showed that the target rice contained... SS3a and SS3b All genes underwent frameshift mutations, resulting in complete loss of function; digestibility and physicochemical quality analysis results showed that... SS3a and SS3b The gene co-knockout significantly increased the resistant starch content and significantly reduced the digestibility of rice, while also exhibiting significant changes in physicochemical qualities, resulting in a marked improvement in its nutritional and health benefits. Applying the method for cultivating high-resistant starch rice and related genetic materials of this invention to rice breeding practices is expected to bring new breakthroughs in the selection of nutritious and healthy new rice varieties.

The present application relates to the field of genetic engineering, in particular to a sgRNA group targeting DYA gene, a primer pair for amplifying target sequence, a plasmid group and application thereof. The sgRNA group designed by the present application edits the sheep DYA gene, and after gene editing, large fragment deletion occurs and causes frame shift mutation, which causes great damage to the protein structure and function encoded by the DYA gene. The experimental results show that different fragment lengths of gene editing can be caused in the target region, the overall editing efficiency of the first exon reaches 76.4%, and the overall editing efficiency of the second exon reaches 84.09%. Editing occurs on both sets of chromosomes in the genome (i.e. homozygote): the homozygote editing efficiency of the first exon is 30.9%, and the homozygote editing efficiency of the second exon is 9.1%. The sgRNA group provided by the present application has important significance in the study of ruminant evolution and related immune mechanisms.

The invention belongs to the technical field of gene engineering, and relates to application of a wheat negative regulation anti-disease gene TaZF10 in resisting gibberellic disease. The invention verifies that the gene TaZF10 negatively regulates the resistance of wheat to gibberellic disease, the protein TaZF10 coded by the gene TaZF10 interacts with gibberellic disease resistant protein TaSnRK1 alpha, and the TaSnRK1 alpha interferes with the stability of the TaZF10 protein. The gene TaZF10 is subjected to frameshift mutation through a CRISPR-Cas9 gene editing technology, so that the protein TaZF10 loses functions, a gibberellic disease resistant material is created, and an effective way and material reserve are provided for wheat disease resistance breeding and disease prevention and control.

The present application relates to BnAHL15 gene and its application in regulating early flowering of plants, especially the application of BnAHL15 gene of rapeseed in regulating early flowering of plants, and belongs to the technical field of biology.The present application firstly designs sgRNA which is specific to BnAHL15 gene, makes oligo dimer of the sgRNA, and constructs a gene editing vector with a Cas9 skeleton, and then introduces the gene editing vector into hypocotyl callus of Brassica napus by means of agrobacterium-mediated genetic transformation technology, and generates seedlings again, under the guidance of sgRNA, Cas9 nuclease edits BnAHL15 gene on the genome, produces a frameshift mutation, causes early termination of gene coding, and phenotype identification finds that the flowering period of homozygous mutant lines of rapeseed is advanced, and the size (yield) of seeds does not change obviously.

A primer for detection of a mutation in a microsatellite contained in a target sequence of a double stranded DNA molecule, wherein the mutation is a frameshift of the microsatellite as compared with the corresponding wild type microsatellite sequence. The primer comprises a region of at least 10 nucleotides that is complementary to the target sequence of the antisense or the sense strand of the DNA molecule containing the microsatellite having a frameshift mutation, except that the primer includes between one and four, or one and three, nucleotides which are mismatched to the target sequence containing the mutation in the microsatellite and which are also mismatched to a corresponding sequence containing the wild type microsatellite.

The application provides a molecular marker for detecting a premature senescence gene of crops and application thereof, and belongs to the technical field of crop breeding. The molecular marker for detecting the premature senescence gene of crops can detect the deletion of four bases AACA in the 11th exon of the coding region of the gene LOC_Os03g31550, so as to form a premature senescence related gene caused by frame shift mutation. Research shows that the rice containing the premature senescence related gene has a stable premature senescence phenotype, the leaf tips of the second leaf and the third leaf are dried and the premature senescence phenotype appears on the 10th day after sowing in a field environment, and the aging lasts to the mature period. The photosynthetic pigment content of mesophyll cells is obviously reduced in the tillering period, the chloroplast structure is changed, the light damage is serious, the photosynthesis is weakened, and the uric acid content is reduced. The application provides a molecular basis for further in-depth research on gene regulation of aging, and provides a reference for cultivating new varieties resistant to premature senescence by using the premature senescence gene.

The invention discloses sgRNA of a targeted F5H gene, a recombinant plasmid, a recombinant bacterium, a gene knockout method and application, and belongs to the technical field of plant genetic engineering and forestry biology. In order to solve the problem that in the prior art, a method for cultivating a new variety of poplar by completely knocking out an F5H gene, the content of lignin S monomer is almost reduced to zero, and the total lignin content and biomass are not influenced, sgRNA capable of targeting the F5H gene is obtained based on multistage screening of bioinformatics; the sgRNA shows the highest homozygous / diallele mutation efficiency in a transformed plant, fragment deletion or frameshift mutation is realized on all three target genes, and the sgRNA, the recombinant plasmid containing the sgRNA and the recombinant agrobacterium can be used for cultivating a new variety of poplar with lignin S monomer deletion and significantly increased G monomer proportion.

An improved promoter and a use thereof. An improvement is to mutate a nucleic acid sequence between −35 region and −10 region in a promoter region into recognition sites for an endonuclease. The improvement can overcome the problem that a strong promoter in a vector based on blue-white screening initiates the transcription or translation of foreign genes and a transcription or translation product might be toxic to a host and cannot be cloned, avoid the deficiency that frameshift mutation of a gene due to a lack of 1-2 bp of the vector at digestion sites results in false positive clones, and eliminate a false negative phenomenon that a plate is rich in blue spots due to a small fragment of foreign DNA and a reading frame of the gene which is unchanged by inserting the foreign DNA.

The invention discloses a method for targeted knockout of a pig PKD2 gene locus and application, and belongs to the technical field of gene editing engineering. A PKD2 gene knockout method is designed on the basis of a CRISPR / Cas9 system, frameshift mutation simulation gene knockout can be achieved, specifically, pX330-E9 targeting plasmids are constructed on the basis of design of specific sgRNA, gene editing is conducted on Bama pig embryo fibroblasts, cell lines with three different mutation types and PKD2 gene knockout are successfully obtained, and the PKD2 gene knockout method has the advantages that the PKD2 gene knockout method is suitable for large-scale popularization and application. The PKD2 gene knockout porcine fibroblast model has important application value in porcine gene editing research and related fields, and particularly provides an important platform for pathogenesis research, treatment method evaluation and new treatment target exploration of the polycystic kidney disease by the PKD2 gene knockout porcine fibroblast model.

Monocot plants heterozygous for centromeric histone 3 (CenH3) and optionally expressing gene editing constructs, for use in inducing haploids of a monocot target plant and optionally pass-through gene editing are provided. The monocot haploid inducer plants are typically composed of diploid plant cells having only one allele encoding a functional CENH3 protein. The diploid plant cells can also include, for example, one CenH3 allele encoding non-functional CENH3 protein. In some embodiments, the allele encoding non-functional CENH3 protein is a frameshift mutation, protein null allele, an RNA null allele, or a combination thereof. The monocot haploid inducer plant can also include gene editing machinery, such as a site-directed nuclease and optionally a guide RNA stably expressed by cells of the monocot plant. Methods of inducing formation of a target haploid monocot plant while optionally simultaneously modifying the target monocot plant's genome are also provided.

The invention discloses a TMPRSS2 gene modified porcine fibroblast cell strain and a set of gene modification system capable of being used for knocking out a porcine TMPRSS2 gene. The gene modification system can be used for preparing a TMPRSS2 gene modified cell strain, the TMPRSS2 gene in the cell strain is knocked out, specifically, a basic group C is added behind a CCGTCGTCGTCAGCAGCAG sequence of the TMPRSS2 gene of each of two chromosomes of the cell strain, double-frame-shift mutation is generated, and the TMPRSS2 gene is not expressed. According to the invention, the cell strain is preserved, and the preservation number is GDMCC No: 67170. A TMPRSS2 gene modified pig can be prepared through the cell strain, the modified pig has remarkable disease resistance to PEDV, and due to the fact that the TMPRSS2 gene is knocked out, the gene modified pig possibly has disease resistance to various porcine coronaviruses, is a broad-spectrum disease-resistant pig, can naturally resist the porcine epidemic diarrhea virus or relieve symptoms after infection, and has a good application prospect. Further, the use of anti-PEDV medicines is greatly reduced, and the medicine residues, the disease-resistant cost and the infection risk are reduced.

The invention discloses a Crispr / Cas9 gene editing vector of a wheat TaPRR73 gene and application of the Crispr / Cas9 gene editing vector, the vector comprises a pBUE414 skeleton, a Cas9 expression cassette and an sgRNA expression cassette, the sgRNA expression cassette sequentially comprises the following elements from upstream to downstream: an expression cassette for regulating and controlling sgRNA1 expression of TaPRR73 by a TaU3 promoter, a terminator OsU3t, an expression cassette for regulating and controlling sgRNA2 expression of TaPRR73 by the TaU3 promoter, and a terminator TaU3t; wherein the sequence of the sgRNA1 is as shown in SEQ ID NO. 1, and the sequence of the sgRNA2 is as shown in SEQ ID NO. 2. According to the invention, the Crispr / Cas9 gene editing vector of TaPRR73 is successfully constructed on the basis of a Crispr / Cas9 gene editing technology, the TaPRR73 gene frame-shift mutation is realized in a wheat material, and an effective method is provided for TaPRR73 gene knockout.

This invention discloses a Mycoplasma caprineis subspecies Mccp NM strain and its applications. Whole-genome alignment of this strain revealed 422 missense mutations, 132 frameshift mutations, and several key insertion / deletion variations compared to the existing vaccine strain C87001. In virulence studies, at a concentration of 1×10⁻⁶... 9 In healthy, susceptible goats, intratracheal injection at a CCU / mL dose resulted in all experimental goats exhibiting typical symptoms of caprine contagious pleuropneumonia. Autopsy revealed pleural effusion and liver-like lesions in the lungs. The inactivated vaccine prepared using this strain demonstrated good safety in goats. Challenge protection tests showed that the vaccine of this invention provided 100% protection against homologous challenge with the NM strain and 80% protection against the heterologous C87002 strain, while commercially available vaccines offered only 40% protection against the NM strain. This invention overcomes the deficiency of existing vaccines in providing insufficient protection against clinical variants, offering an effective technical reserve and candidate vaccine for addressing immunization failure caused by Mccp antigen mutations.

The application relates to a pigeon TYRP1 Mutant of a gene and application thereof, and belongs to the technical field of molecular biology. The nucleotide sequence of the mutant is shown as SEQ ID NO. 1. The application utilizes TYRP1 A frame shift mutation c.891delT (p.T297fs) of the gene has a double phenotype effect, so that it is possible to use eye color as a gender identification marker of white-feathered pigeons, and a bottleneck in application of feather color self-alternation technology in mainstream white-feathered breeds is broken.

The application discloses a kind of gene molecular marker assisted screening fresh soybean high-quality germplasm rapid creation method, it is related to genetic engineering technical field.For GmLOX1 gene, GmLOX2 gene, GmLOX3 gene, GmBadh1 gene and GmBadh2 gene frameshift mutation editing site designs specific primer, the specific primer is only specific amplification editing genotype, does not amplify wild genotype.Can assist in screening no soybean smell, with the fragrance of fresh soybean, also can distinguish editing genotype soybean and wild genotype soybean.The method is simple in operation, screening efficiency is high, and screening cycle is short.

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of an AIL1 gene in regulation and control of soybean internode spacing and cultivation of close-planting high-yield soybean varieties. The research on the soybean AIL1 gene finds that the variation of the soybean AIL1 gene can cause the change of the internode cell length and influence the internode spacing, specifically, the frameshift mutation of the AIL1 gene can cause the shortening of the soybean internode spacing; therefore, an AIL1 soybean mutant is further created by utilizing a gene editing technology, a plurality of natural excellent allelic variation mutants are identified in a large number of soybean materials, and the excellent allelic variation mutants are backcrossed to a high-yield soybean variety which is not resistant to close planting. The obtained backcross material is stable in internode spacing under different close planting conditions, and the yield is slightly influenced by density, so that the AIL1 gene can be used for cultivating high-yield close planting soybean varieties, and the development of the soybean industry is promoted.

This invention discloses an α-rhamnosylase mutant and its applications, belonging to the field of microbial engineering technology. The α-rhamnosylase mutant disclosed in this invention has the amino acid sequence shown in SEQ ID NO.2, which consists of a total of 268 amino acid residue point mutations and two frameshift mutations adding 14 amino acid residues based on the parental amino acid sequence of α-rhamnosylase SEQ ID NO.1. Results show that the parental α-rhamnosylase and the α-rhamnosylase IgRha M24-5 mutant enzyme have conversion rates of 72.84% and 99.76% for astragalus root C, respectively; and conversion rates of 90.33% and 99.81% for rutin, respectively. This indicates that the α-rhamnosylase mutant has good application prospects in the selective biotransformation of flavonoid glycosides to prepare derhamnosylflavones under high-temperature conditions.

The invention relates to the field of cancer, in particular breast cancer. In particular it relates to the field of immune system directed approaches for tumor reduction and control. Some aspects of the invention relate to vaccines, vaccinations and other means of stimulating an antigen specific immune response against a tumor in individuals. Such vaccines comprise neoantigens resulting from frameshift mutations that bring out-of-frame sequences of the GATA3, CDH1, MAP3K1, RUNX1, and TP53 genes in-frame. Such vaccines are also useful for ‘off the shelf’ use.

The invention provides a gene BBX15 for regulating and controlling the heading stage of millet and application, and belongs to the technical field of functional genes. The invention provides a gene BBX15 capable of being used for regulating and controlling the heading stage of millet. The gene BBX15 is subjected to frame-shift mutation by using a gene editing technology, so that the coded protein is terminated in advance, and a mutant material is constructed. Compared with a wild type, the mutant material has the advantage that the heading period is obviously advanced. Therefore, the gene BBX15 provides a novel regulation and control way for regulating and controlling the heading period characters of the plants, and has important value for plant character improvement and breeding practice.

The invention relates to a preparation method of a gRNA and OPG gene knockout non-human animal model of a targeted non-human animal OPG gene. The preparation method of the OPG gene knockout non-human animal model comprises the following steps: a non-human animal OPG gene is destroyed by using a gene editing technology, and gRNA for targeting the non-human animal OPG gene comprises one or two of SEQ ID NO: 1 and / or SEQ ID NO: 2. The gRNA specifically targeting the OPG gene of the non-human animal is designed, and the OPG gene is knocked out by using Cas9 protein to cause frameshift mutation, so that the purpose of performing gene knockout on the non-human animal is achieved. The method for modifying the OPG gene of the non-human animal is simple and easy to implement and short in period, the OPG model constructed by the method provides a basis for researching the physiological mechanism and the molecular mechanism of the OPG gene, and meanwhile, an important tool is provided for researching the pathogenesis of osteoporosis and developing medicines.

The present disclosure provides a lentivirus-like particle for treating Huntington's disease (HD). The lentivirus-like particle VLP-HD is produced by encapsulating an RNP complex including a Cas9 protein and an HTT gene-targeted gRNA with a virus-like particle (VLP) vector. After infecting cells, VLP-HD releases Cas9:gRNA RNP, which can efficiently achieve the targeted knockout of the mutant HTT gene. The present disclosure can prevent the production of the mutant HTT protein with PolyQ at the source, thereby achieving the etiological therapy for HD. VLP-HD can be either single-target VLP that delivers a single CRISPR / Cas9 system to knock out the mutant gene through a frameshift mutation of the mHTT gene, or dual-target VLP that delivers two CRISPR / Cas9 systems to delete disease-causing CAG trinucleotide repeats in the mHTT gene, thereby completely avoiding the production of PolyQ.