35 results about "Frameshift mutation" patented technology

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 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.

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.

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 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.

The invention provides a retina photoreceptive neuronal lesion model based on fads2 and a preparation method thereof, and relates to the technical field of gene engineering. Wherein the sgRNA comprises a guide sequence which can be specifically combined with a nucleotide sequence as shown in SEQ ID NO.4 in the fads2 gene. The sgRNA can be used for constructing an animal individual highly similar to human retinitis pigmentosa disease through precise targeting and causing specific frame-shift mutation of the zebra fish fads2 gene. As a key tool for preparing the model, the method provides reliable, economic and efficient support for deeply researching the pathological mechanism of diseases and accelerating drug screening.

This invention relates to the field of genetic engineering technology, disclosing a method for constructing a plasmid for knocking out the FOSB gene, the plasmid constructed by the above method, and the application of the plasmid in preparing transfection reagents for knocking out the FOSB gene in cell lines; it also discloses a FOSB gene knockout method based on the CRISPR-Cas9 system, FOSB gene knockout cells, and their applications. This invention provides a method for designing dual sgRNAs targeting exons 2 and 3 of the FOSB gene, overcoming technical bottlenecks such as polyploidy / alloidy in U87-MG cells, high DNA repair capacity, and easy phenotype recovery, significantly improving knockout efficiency, causing frameshift mutations, resulting in complete loss of FOSB protein function, and constructing a knockout vector based on the PX459 plasmid, adapted to U87-MG cells, solving the problem of low conventional transfection efficiency; and successfully constructing the FOSB gene knockout vector. ∆ / ∆ The U87 cell line can be stably passaged for a long time, has a stable genetic background, and does not lose the knockout phenotype, providing a standardized and reproducible experimental platform for FOSB gene function research and glioma mechanism exploration.

The invention discloses a construction method and application of a DFNB1 type deafness mouse model with GJB2 gene frameshift mutation. The construction method comprises the following steps: (1) designing two sgRNA plasmids on a No.2 exon of a mouse Gjb2 gene; (2) screening out mouse embryonic stem cells of which the Gjb2 gene carries a c.299300delAT mutation site; (3) forming a recombinant embryo, transplanting the recombinant embryo into the uterus of the pregnant female mouse, and waiting for pregnancy and farrowing to obtain an F0-generation mouse; and (4) screening the F0 generation mice in the step (3) to obtain the mouse model carrying the c.299300delAT homozygous point mutation of the GJB2 gene. The method has the advantages that the problem of mouse embryo metaphase energy supply disorder lethal caused by Gjb2 homozygous mutation is solved, the embryo survival rate is increased, and the bottleneck of embryo lethal in model construction of a traditional gene editing technology is broken through.

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

An information processing apparatus including a processor, in which the processor is configured to: use a first prediction model that outputs a first prediction evaluation result indicating whether or not a candidate substance has mutagenicity related to a base pair substitution mutation, and a second prediction model that outputs a second prediction evaluation result indicating whether or not the candidate substance has mutagenicity related to a frameshift mutation; acquire candidate substance information related to the candidate substance; input the candidate substance information or information based on the candidate substance information to the first prediction model and the second prediction model, and output the first prediction evaluation result and the second prediction evaluation result from the first prediction model and the second prediction model; and present prediction information corresponding to the first prediction evaluation result and the second prediction evaluation result to a user.

The invention belongs to the field of molecular biology, provides a functional molecular marker of a major gene AhARF2 for regulating and controlling the oil content of peanut seed kernels and application of the functional molecular marker, discloses a major site qSOCA08-3 for regulating and controlling the oil content of the peanut seed kernels, and explains phenotypic variation of 14.91%-17.31%. The qSOCA08-3 is finely positioned to a physical interval of 349.6 kb between the Tif2. A08.49681404 and the Tif2. A08.50031016, and a candidate gene of the qSOCA08-3 is confirmed to be AhARF2. The qSOCA08-3 has the advantages that the qSOCA08-3 can be used for identifying the The AhARF2 coding sequence shows 275bp deletion between parents Jianhua No.1 and W191 and is accompanied by 7bp replacement, so that the code shift mutation of the coding sequence is caused, and the protein translation is terminated in advance. According to the invention, the KASP molecular marker is developed on the basis of a mutant sequence, the association between the KASP molecular marker and the oil content of the seed kernel is successfully verified in a Yuhua 15 * ST001 recombinant inbred line population, and the important value of the marker in the marker-assisted selection breeding of the high oil content of the seed kernel of the peanut is verified.