1070 results about "Endonuclease" patented technology

The invention relates to the improvement of endonuclease-based antimicrobials by blocking DNA repair of double-strand break(s) (DSB(s)) in prokaryotic cells. In this respect, the invention especially concerns a method involving blocking DNA repair after a nucleic acid has been submitted to DSB, in particular by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated programmable double-strand endonuclease. The invention particularly relates to the use of an exogenous molecule that inhibits DNA repair, preferably a protein that binds to the ends of the double-stranded break to block DSB repair. The invention also relates to vectors, particularly phagemids and plasmids, comprising nucleic acids encoding nucleases and Gam proteins, and a pharmaceutical composition and a product containing these vectors and their application.

To provide a method for detecting the presence of multiple specific nucleic acid sequences or single nucleotide polymorphisms in a temperature-dependent fashion.SOLUTION: Provided is a method, in which an invasive oligonucleotide and a primary probe oligo (a 5' flap oligonucleotide) hybridize to a target strand nucleic acid to form an invasive cleavage structure cleavable by a FEN-1 endonuclease to release a cleaved 5' flap (primary cleaved flap); in a secondary reaction, the primary cleaved flap from the primary reaction hybridizes to a FRET cassette 1, a hairpin oligonucleotide labeled with a fluorescent dye and a quencher molecule, to form a second invasive cleavage structure that is cleavable by a FEN-1 endonuclease at a site between the fluorophore and a quencher; and cleavage of FRET cassette 1 separates the quencher from the fluorophore, such that fluorescence signal from the fluorophore can be detected.SELECTED DRAWING: Figure 1

The present invention provides a method and a kit for detecting a target nucleic acid to be detected through a melting curve. The method includes designing a first primer, a second primer and a detection probe for the target nucleic acid sequence to be detected, performing PCR amplification in a PCR amplification system containing the first primer, the second primer, the detection probe, the sample to be detected, a restriction endonuclease and a DNA polymerase, generating a reporter primer that is complementary to and pairs with the detection probe to form a double-stranded product, obtaining the melting curve of the double-stranded product, and the melting curve of the double-stranded product is the melting curve corresponding to the target nucleic acid to be detected. The method of the present invention is a non-target-dependent melting curve method, and the melting point (T m value) of each double-stranded body can be calculated in advance, solving the problems of melting curve peak shift and easy misjudgment caused by easy mutation of the target nucleic acid sequence to be detected.

The present invention discloses a Gs12-7 endonuclease variant and a gene editing system mediated thereby. Specifically, by means of a rational mutation strategy, two Gs12-7 mutants were constructed and compared, and it was found that after the 157th amino acid of the Gs12-7 endonuclease was mutated from Glu to Arg, the activity of this gene editing enzyme could be significantly improved, and this variant was designated as Gs12-7MAX. The present invention provides an efficient gene editing technology mediated by the CRISPR-Gs12-7MAX system, which has broad application prospects in the field of genome site-directed modification.

Provided are a double-stranded oligonucleotide agent and uses. The double-stranded oligonucleotide agent comprises a specific cleavage site for an endonuclease, wherein the cleaved double-stranded oligonucleotide agent is capable of inhibiting expression of the target gene via RNA interference (RNAi).

The invention relates to antarctic krill peptide with umami and an antarctic krill peptide mixture as well as a preparation method and application thereof. Specifically, the amino acid sequence of the euphausia superba peptide is shown as any one of SEQ ID NO: 1-5. The euphausia superba peptide mixture with delicate flavor is obtained by carrying out enzyme digestion treatment on an euphausia superba peptide solution by using incision enzyme, excision enzyme and freshness enhancing enzyme and harvesting supernate. Molecular docking simulation shows that the euphausia superba peptide disclosed by the invention can be combined with a delicate flavor receptor T1R1 / T1R3. The Antarctic krill peptide and the Antarctic krill peptide mixture can be used for development of umami seasonings, and a new idea is provided for development of marine seasonings.

Methods and compositions for modulating a target genome and stable integration of a transgene of interest into the genome of a cell are disclosed. Specifically, provided herein comprises compositions comprising an RNA molecule comprising a reverse complement sequence comprising an exogenous sequence and homology arms, an RNA molecule comprising an endonuclease, and one or more guide RNAs for specific genomic integration of the exogenous sequence.

Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity of RNA-programmable endonucleases, such as Cas9, or for controlling the activity of proteins comprising a Cas9 variant fused to a functional effector domain, such as a nuclease, nickase, recombinase, deaminase, transcriptional activator, transcriptional repressor, or epigenetic modifying domain. For example, the inventive proteins provided comprise a ligand-dependent intein, the presence of which inhibits one or more activities of the protein (e.g., gRNA binding, enzymatic activity, target DNA binding). The binding of a ligand to the intein results in self-excision of the intein, restoring the activity of the protein.

This invention relates to recombinant nucleic constructs comprising a DNA binding domain, an endonuclease and a reverse transcriptase and methods of use thereof for modifying nucleic acids in plants.

The invention discloses a novel PDRN, a supramolecular liposome containing the PDRN as well as a preparation method and application of the supramolecular liposome. The preparation method of the novel PDRN comprises the following steps: (1) mixing a tissue raw material and PBS, crushing, carrying out enzymolysis, adding SDS, a Tween20 solution, polyethylene glycol octylphenol ether, a TE buffer solution and NaCl, stirring, centrifuging, mixing a supernatant with absolute ethyl alcohol, and standing to obtain a DNA crude extract; (2) carrying out breaking shearing treatment by using an ultrasonic breaking instrument; and (3) carrying out enzymolysis treatment by using restriction endonuclease and deoxyribonuclease, and the like. The PDRN supramolecular liposome is obtained by wrapping the PDRN with fatty acid extracted from marine microalgae. The fish testis are used as the raw material source, the selection range of the raw materials is widened, the preparation cost is reduced, and the organic reagent used in the preparation method is ethyl alcohol which is easy to remove and harmless.

The invention relates to an endonuclease S2 mutant, a preparation method of the endonuclease S2 mutant and application of the endonuclease S2 mutant in antibody glycosylation transformation. The glycosidase S2 mutant comprises a carbohydrate binding module (CBM) defined by M677-D843 in SEQ ID NO: 1, and the amino acid sequence of the glycosidase S2 mutant comprises SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

Provided herein are novel TnpB polypeptides having RNA guided endonuclease activity. Also provided herein is a gene editing system comprising the TnpB polypeptide of the invention or a disarmed variant thereof, or a fusion polypeptide comprising the same, and a method of gene editing using the TnpB polypeptide of the invention or a disarmed variant thereof, or the fusion polypeptide or the gene editing system.

The invention belongs to the technical field of fish genetic identification, discloses a multi-site large yellow croaker wild domestication population identification system based on an agarose gel electrophoresis technology, and particularly discloses a group of InDel molecular markers for large yellow croaker wild and domestication population identification, and the InDel molecular markers comprise InDel11-5053756, InDel20-14004876, InDel3-28345321 and InDel21-17327392. The InDel molecular marker detection primer is used for amplification to obtain a PCR product, agarose gel can be used for detecting strip polymorphism, extra restriction enzyme digestion or complicated polyacrylamide gel electrophoresis operation is not needed, and identification of large yellow croaker wild and domesticated populations can be rapidly and accurately achieved.

The invention discloses a Vinck Cellulomicrobe and application thereof in degradation of corn straw and improvement of soil fertility, and belongs to the field of agricultural microbial technology and solid waste resource utilization. The bacterial strain can secrete a plurality of hydrolase and oxidase including AA10 family lytic polysaccharide monooxygenase, AA2 peroxidase, GH3 and GH1 beta-glucosidase, GH16 / GH17 beta-glucosan endonuclease and AA1 multi-copper oxidase. The bacterial strain has the advantages that the bacterial strain can secrete a plurality of hydrolase and oxidase; fermentation liquor is applied to soil according to the straw mass ratio of 1: 5, and the corn straw degradation rate can be increased under the condition that the mass ratio of soil to straw is 100: 1. Meanwhile, the microbial agent can synergistically improve soil nutrients and increase the contents of rapidly available phosphorus, rapidly available potassium, alkali-hydrolyzable nitrogen, soluble organic carbon and total phosphorus in soil. According to the microbial agent, a straw lignocellulose structure is effectively cracked through a multi-enzyme system synergistic effect, and the dual effects of accelerating straw degradation and improving soil fertility are achieved.

The invention discloses a preparation method of high-purity circular RNA (Ribonucleic Acid). The preparation method comprises the following steps: constructing a cyclization skeleton sequence with self-splicing activity; the cyclization skeleton sequence comprises the following modular elements from the 5'end to the 3 'end: a T7 promoter, a 5'-terminal homologous fragment, a 3 'intron-exon fragment, a 5'-internal homologous fragment, a 5 '-terminal spacer sequence, an IRES sequence, a target fragment, a 3'-terminal spacer sequence, a 3 '-internal homologous fragment, an exon fragment-5' intron, a 3 '-terminal homologous fragment and an Xba I endonuclease site; inserting the cyclized skeleton sequence into a pUC57 vector, and carrying out plasmid amplification; an Xba I incision enzyme linearization vector is utilized to obtain an in-vitro amplification template; the method comprises the following steps: carrying out an in-vitro amplification reaction by using dsRNA-free T7RNA polymerase to obtain dsRNA-free precursor RNA; cyclizing the precursor RNA to obtain crude circular RNA; treating the crude circular RNA by utilizing poly (A) or poly (U) polymerase, and adding poly (A) or poly (U) long tails to linear RNA in the crude circular RNA; and purifying by using an affinity chromatography column to obtain the high-purity circular RNA.

The invention relates to the technical field of genetic engineering, in particular to a preparation method of a zebrafish trmt61a maternal zygote mutant, which comprises the following steps: co-injecting sgRNA and Cas9 protein of a targeted zebrafish trmt61a gene into a one-cell stage embryo of wild type zebrafish to obtain a trmt61a heterozygote mutant; the method comprises the following steps: constructing pGGDestISceIEG-4sgRNA plasmids, and injecting the pGGDestISceIEG-4sgRNA plasmids, an incision enzyme and a reaction buffer solution into a one-cell stage embryo of Tg (zpc: zcas9) transgenic zebrafish to obtain an adult zebrafish F0 generation; and hybridizing the female fish of the adult zebra fish F0 generation with the male fish of the trmt61a heterozygote mutant to obtain the zebra fish trmt61a maternal zygote mutant. The method breaks through the limitation of trmt61a gene homozygous mutation death, and realizes rapid preparation of the maternal zygote mutant.

Methods to edit genes by administering a chimeric nuclease to a cell or organism without the use of a viral vector.

Provided herein are modified Staphylococcus aureus Cas9 nucleases and methods of using modified Staphylococcus aureus Cas9 nucleases. Also provided herein are chimeric nucleases comprising an I-Tevl nuclease domain, and a Staphylococcus aureus Cas9 and methods of using the chimeric nuclease. Also provided herein are methods to edit genes by administering a chimeric nuclease to a cell or organism without the use of a viral vector.

The invention discloses a molecular marker linked with yellow peel of bitter gourd and application of the molecular marker. It is found through analysis that mutation of a base G at the 199th site of SEQ ID NO.1 into A is a reason for formation of bitter gourd yellow peel. A specific primer is designed aiming at the mutation site, a bitter gourd genome is amplified by utilizing the primer, a PCR amplification product is subjected to incision enzyme XhoI enzyme digestion, and the bitter gourd variety with yellow peel can be identified according to the size of an enzyme digestion product. The method can accurately identify the peel color in the early growth stage of the bitter gourd, improves the breeding efficiency of the yellow peel variety of the bitter gourd, and has the advantages of high accuracy, simplicity and convenience in operation and the like.

The invention relates to a polygene methylation detection kit for liver cancer, a detection system and application thereof, and aims to provide a marker combination for early liver cancer screening. The kit is used for detecting the methylation state of one or more of 114 genes of BDH1, CPAMD8, DAB2IP, DLEC1, HOXA1, MIXL1, OTX1, PCDH8, RNF135, SIX2, SPINT2, TACSTD2, VIM and ZAR1, and is combined with restriction enzyme digestion and fluorescent quantitative PCR (Polymerase Chain Reaction) technologies, so that the early diagnosis of the liver cancer is realized. The kit and the system provided by the invention have high accuracy, specificity and early diagnosis capability, and experiments prove that the detection method can effectively distinguish liver cancer tissues from para-carcinoma tissues, and has relatively high clinical application value. The method is not only suitable for early screening of liver cancer, but also can provide a scientific basis for early treatment of liver cancer patients.

The invention discloses a kit for detecting miRNAs of multiple extracellular vesicles based on a TtAgo-driven rolling circle amplification technology and application of the kit, a platform combines TtAgo protein and RCA reaction, cooperates with endonuclease activity and DNA polymerase activity of TtAgo, accurately controls reaction kinetic balance between temperature and enzyme activity, not only does not damage the cutting activity of TtAgo, but also can detect miRNAs of multiple extracellular vesicles. The method provided by the invention can be used for detecting multiple extracellular vesicles miRNAs, ensures strong polymerization amplification performance, creates a strong one-step single-tube experiment, greatly simplifies the experimental process, shortens the detection time, realizes ultra-sensitive and specific detection of nucleic acid markers, and can be used for detecting multiple extracellular vesicles miRNAs.

Provided herein are Cas endonucleases (and functional fragments, functional variants, and domains thereof), nucleic acid molecules encoding the same, and systems comprising the same. The disclosure further relates to methods of utilizing the Cas endonucleases (or nucleic acid molecules encoding the same), including, e.g., in methods of editing a nucleic acid molecule (e.g., a gene) and methods of treating diseases (e.g., genetic diseases).

Methods and compositions for modulating a target genome and stable integration of a transgene of interest into the genome of a cell are disclosed. Specifically, provided herein comprises compositions comprising an RNA molecule comprising a reverse complement sequence comprising an exogenous sequence and homology arms, an RNA molecule comprising an endonuclease, and one or more guide RNAs for specific genomic integration of the exogenous sequence.

The invention discloses a novel application of emodin as an endonuclease inhibitor. The emodin inhibits replication and transmission of viruses by inhibiting the activity of NSP15 ribonuclease, and recovers host interferon reaction so as to enhance the antiviral effect. The application has the advantages that the action target of the emodin is defined, the effectiveness of the emodin in the aspect of preventing and treating coronavirus infection is further revealed, and the broad-spectrum antiviral potential of the emodin is embodied through the link of playing the antiviral effect by inhibiting the virus replication link; the researches provide candidate drugs and scientific basis for research and development of novel broad-spectrum anti-coronavirus drugs, and promote clinical application of the anti-virus traditional Chinese medicines.

The invention relates to pichia pastoris engineering bacteria and a preparation method thereof, and a method for producing cellooligosaccharide / disaccharide by using corn straws or wheat straws, and belongs to the technical field of genetic engineering. A cellulose incision enzyme gene and a cellulose excision enzyme gene are transferred into pichia pastoris engineering bacteria. The preparation method comprises the following steps: respectively inserting a cellulose incision enzyme gene and a cellulose excision enzyme gene into plasmids to construct expression vectors; transferring the expression vector into a competent cell of pichia pastoris GS115; and screening and identifying to obtain successfully transformed pichia pastoris engineering bacteria. According to the present invention, the pichia pastoris heterologous expression cellulose incision enzyme and cellulose excision enzyme are adopted to construct the pichia pastoris engineering strain capable of degrading the plant straw into the cellooligosaccharide / disaccharide, such that the reliable chassis strain is provided for the production of the starch from the cellulose;

The present disclosure provides one or more engineered gene effectors and systems, compositions, and methods of use thereof, wherein the one or more engineered gene effectors can be used to effect regulation of a target gene in a cell (e.g., an endogenous target gene in a cell). The one or more engineered gene effectors can be operatively coupled to a heterologous endonuclease, such as a CRISPR / Cas protein.

According to the chlorella pyrenoidosa high-F-value peptide and the preparation method thereof, parameters in the enzymolysis and adsorption processes are strictly controlled, and the enzymolysis of the incision enzyme and the excision enzyme not only can efficiently hydrolyze chlorella protein and generate more peptide fragments with biological activity, but also can be used for preparing the chlorella pyrenoidosa high-F-value peptide. And the characteristics of high industrial production cost and poor stability caused by wide enzyme cutting sites are solved. The prepared chlorella pyrenoidosa high-F-value peptide reaches 36.90 + / -8.18, the F value of the chlorella pyrenoidosa high-F-value peptide is increased by 12.72 times compared with the chlorella pyrenoidosa raw material (2.90 + / -0.07), and in addition, the molecular weight of the high-F-value oligopeptide is 1t; peptides with the molecular weight of 1000 Da account for 95-99.40%, the characteristics of oligopeptides are met, the molecular weight is small, and digestion and absorption are easy. The prepared high-F-value oligopeptide can accelerate metabolism of ethyl alcohol in the liver and protect the liver from being damaged by the ethyl alcohol, and has the effects of dispelling the effects of alcohol and protecting the liver.

The present disclosure provides for endonuclease enzymes as well as methods of using such enzymes or variants thereof.