696 results about "Base J" patented technology

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of Cas9 and nucleic acid editing proteins or protein domains, e.g., deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 and nucleic acid editing proteins or domains, are provided.

The disclosure provides, for example, double stranded DNA (dsDNA) molecules comprising one or more chemically modified nucleobases. In some embodiments, the dsDNA molecule is circular and comprises a first strand and a second strand, wherein the first strand comprises one or more chemically modified nucleobases, and the second strand is free of chemically modified nucleobases. In some embodiments, the dsDNA molecule comprises a promoter sequence and an effector sequence that encodes an effector.

The invention belongs to the technical field of genes, and discloses a method for efficiently expressing a foreign protein based on an NC048604-1 site in a CHO cell genome. A site for stably expressing protein in the CHO cell genome is located in the 94142000 to 94148000 basic group range of the CHO cell genome NC048604-1, and the nucleotide sequence of the site is as shown in SEQ ID NO: 1. According to the invention, different protein genes are introduced at fixed positions in a CHO cell genome, and stable expression is carried out.

The invention discloses a method for identifying duck palm weight and related molecular marker application thereof, and relates to the technical field of molecular marker-assisted selection, a primer pair is used for amplifying a DNA fragment containing a 63823029th base polymorphic site from the 5'terminal on a fourth chromosome of a duck reference genome IASAASPekinDuckT2T, and the primer pair is used for amplifying a DNA fragment containing the 63823029th base polymorphic site from the 5 '-terminal on the fourth chromosome of the duck reference genome IASAASPekinDuckT2T. The duck reference genome IAASAASPekinDuckT2T is a duck reference genome sequence in a GenBank database, and the duck reference genome sequence is a duck reference genome sequence in the GenBank database; the duck is a Chinese and new white feather meat duck, and the primer pair consists of a DNA (deoxyribonucleic acid) molecule as shown in SEQ ID No.2 and a DNA molecule as shown in SEQ ID No.3. According to the method for identifying the duck foot weight and the application of the related molecular marker, the genotype of the duck can be judged by detecting the genome DNA of the duck, early living screening of the duck foot weight character is achieved, phenotype determination after slaughtering is not needed, the breeding period is remarkably shortened, and the breeding cost is reduced.

The invention relates to the field of gene engineering. In particular, the present invention relates to cytosine deaminases and their use in base editing. More specifically, the invention relates to a method for screening and identifying a deaminase, a base editing system based on a newly identified cytosine deaminase, a method for editing a target sequence in a genome of an organism (such as a plant) by using the base editing system, and a method for screening and identifying the target sequence. As well as genetically modified organisms (e.g., plants) and progeny thereof produced by the method.

The application discloses a SNP molecular marker for identifying the genetic sex of Procambarus clarkii, wherein the nucleotide sequence of the SNP molecular marker is shown as SEQ ID No. 1, and a male individual is shown as GG homozygote at the 43th base position of the nucleotide sequence of the SNP molecular marker, and a female individual is shown as GA heterozygote at the 43th base position of the nucleotide sequence of the SNP marker. The application further discloses primers and kits for detecting the SNP molecular marker, and application of the reagents, primers and kits for detecting the SNP molecular marker in genetic sex identification of the Procambarus clarkii. The SNP marker is closely related to the genetic sex, and can be used for conveniently and quickly identifying the genetic sex of the Procambarus clarkii in the process of family breeding and unisexual breeding of the Procambarus clarkii, and provides a theoretical basis for identification of sex-reversed individuals in the breeding process.

A composition of matter includes an antisense phosphorodiamidate morpholino oligomer (MO) that includes an MO base sequence. The MO base sequence is arranged to bind a corresponding complementary base sequence of messenger RNA (mRNA) transcribed from one or more genes for the beta subunit of human chorionic gonadotropin (hCG-β). An inventive method, for suppressing intracellular synthesis of a beta subunit of human chorionic gonadotropin (hCG-β), includes introducing such an MO into one or more cells.

The invention relates to the technical field of gene editing, in particular to a Cas9 protein mutant and application thereof. According to the present invention, the mutation is introduced at the position far away from the catalytic site to induce the Cas9 protein conformation change so as to obtain the new Cas9 protein mutant, only the Cas9-sgRNA target chain is cut, and the Cas9-sgRNA non-target chain is not cut, such that the new Cas9 protein mutant can be used for constructing the novel base editor, and the high-efficiency base editing can be performed on the mammalian cell genome. The Cas9 protein mutant disclosed by the invention is fused with a mutant TX with DNA binding deletion of 3 '-> 5' exonuclease TREX2, so that the gene editing efficiency of a 3'terminal generated by paired single-nick enzyme cutting can be greatly improved, and the safety of the Cas9 protein mutant is maintained.

The invention relates to a DNA framework structure addressable enzyme ligation method. Specifically, the invention provides a method for accurately regulating and controlling the specific position of an enzyme on a DNA frame structure through an enzyme modification and base complementary pairing principle, and provides a frame nucleic acid with a capture segment and an enzyme with a connecting chain, and the frame nucleic acid with the capture segment and the enzyme with the connecting chain are subjected to base complementary pairing through the connecting chain and the capture segment. Therefore, the enzyme is connected to a predetermined binding site of the framework nucleic acid, and accurate regulation and control of the connection position, number spacing and the like of the enzyme on the framework nucleic acid are realized. The connection method provided by the invention can realize addressable and accurate enzyme position regulation and control on the DNA nano structure, can widen site-specific modification of the enzyme on the DNA frame structure, and has important significance for developing site-specific and high-throughput enzymatic reaction.

Compositions and methods comprising a deaminase for targeted editing of nucleic acids are provided. Also provided are compositions and methods for localizing a heterologous polypeptide to a target DNA molecule, and compositions and methods for targeted editing of nucleic acids. Fusion proteins comprising an RNA-guided nuclease (RGN) and at least one heterologous polypeptide inserted therein are provided, as well as fusion proteins comprising a DNA binding polypeptide and a deaminase. The heterologous polypeptide may be a pilot editing polypeptide or a base editing polypeptide. Compositions also include nucleic acid molecules encoding a deaminase or fusion protein. Vectors and host cells comprising the nucleic acid molecules encoding the deaminase or fusion protein are also provided.

Embodiments are directed to N3-kethoxal reagents and derivatives thereof, and related methods that allow fast and reversible labeling of single-stranded nucleic acids in live cells. By way of example, one aspect is directed to a process for reversible labeling of single-stranded guanine bases in live cells, which results in an effective in vivo method for transcriptome-wide RNA secondary structure mapping and RNA G-quadruplex prediction.

The invention discloses a Cg12n-v4.6 nuclease. The amino acid sequence of the nuclease is as shown in SEQ ID No. 12. The invention also discloses a preparation method of the nuclease. The Cg12n-v4.6 nuclease mutant with increased activity and improved editing efficiency is developed, and the editing efficiency of the Cg12n-v4.6 nuclease mutant is up to 70% or above. Meanwhile, through engineering modification of corresponding sgRNA of Cas12n, the size of the sgRNA skeleton is reduced. An enhanced Cas12n gene editing tool, namely an eCg12n gene editing system, is developed by combining an engineered Cg12n-v4.6 nuclease mutant and a simplified sgRNA skeleton. Meanwhile, by fusing deaminase, a compact cytosine base editor and a compact adenine base editor are constructed, and the cytosine base editor is applied to edit and induce disease genes to generate early termination codons, so that safe knockout of the genes is realized. Compared with an original wild type editing system, the Cg12n-v4.6 nuclease mutant and a compact eCg12n gene editing system derived from the Cg12n-v4.6 nuclease mutant have the advantages that the editing efficiency is improved, the system size is further simplified, and the Cg12n-v4.6 nuclease mutant and the compact eCg12n gene editing system derived from the Cg12n-v4.6 nuclease mutant are more suitable for gene editing application.

The invention relates to a group of specific amplification primers for mitochondrial genes of procambarus clarkia and application thereof, and belongs to the technical field of molecular biology, and the mitochondrial genes of the procambarus clarkia comprise cytochrome c oxidase I subunit genes and cytochrome b genes. The specific amplification primer of the CO I gene comprises a CO I gene upstream primer and a CO I gene downstream primer, wherein the base sequences of the CO I gene upstream primer and the CO I gene downstream primer are respectively shown as SEQ NO.1 and SEQ NO.2; the specific amplification primer of the Cytb gene comprises a Cytb gene upstream primer and a Cytb gene downstream primer of which the base sequences are respectively as shown in SEQ NO.3 and SEQ NO.4. The invention further discloses a kit for detecting the Cytb gene. According to the amplification method of the mitochondrial gene of the procambarus clarkia, PCR (Polymerase Chain Reaction) amplification is carried out on a DNA (Deoxyribonucleic Acid) template solution of the procambarus clarkia by adopting amplification primers of a mitochondrial COI gene and a Cytb gene of the procambarus clarkia. The procambarus clarkia mitochondrial gene specific amplification primer has high specificity, a single target gene can be stably amplified, the length of the obtained procambarus clarkia mitochondrial COI gene segment reaches about 1000 bp, and the length of the obtained procambarus clarkia mitochondrial Cytb gene segment reaches about 800 bp.

The invention discloses a method for light regulation and control of Cas12a crRNA activity and an application of the Cas12a crRNA activity. Light-caged crRNA is designed according to a target nucleic acid sequence, and is selected from any one or two of the following: (1) complete crRNA: carrying out single-base light-caged modification on nucleotide at a third site or a fourth site at a 5'end of a crRNA fixed region; (2) splitting the crRNA: splitting the crRNA from a stem loop of the fixed region and containing two sequences, the first sequence is nucleotide from the first site to the tenth site at the 5'end of the fixed region of the crRNA, and the nucleotide at the third site or the fourth site at the 5 'end of the fixed region of the crRNA is subjected to single-base light cage modification; the second sequence is the remaining part of the fixed region and the spacer region after the 11th site to the 14th site at the 5'end of the crRNA fixed region are removed. The light-caged crRNA can be used for detecting a nucleic acid sample to be detected by a one-tube method, and the detection sensitivity is high.

The invention discloses a divalent nucleic acid aptamer fluorescence detection method based on an exonuclease I auxiliary target circulation strategy, and belongs to the field of analysis and detection.The divalent nucleic acid aptamer is a repetitive sequence composed of two monovalent nucleic acid aptamers and can form double-stranded DNA through base complementary pairing with complementary strand cDNA of the divalent nucleic acid aptamer, and the divalent nucleic acid aptamer can be used for fluorescence detection of the divalent nucleic acid aptamer. The method comprises the following steps: taking a bivalent nucleic acid aptamer as a template, forming a DNA silver nanocluster (DNA-AgNCs) under the action of silver nitrate and sodium borohydride, and when the target okadaic acid exists, combining the bivalent nucleic acid aptamer with the target, so that cDNA can be replaced from double-stranded DNA by the target. Exonuclease I is added to perform enzyme digestion on the cDNA and the divalent nucleic acid aptamer, so that the fluorescence intensity of the DNA-AgNCs can be rapidly reduced. The content of the okadaic acid can be judged by detecting the change of the fluorescence intensity. The invention provides a simple, convenient, rapid, high-sensitivity and high-specificity detection method for okadaic acid detection based on a divalent nucleic acid aptamer for the first time.

Provided is a reverse prime editing system, which is used for achieving site-specific and precise small-fragment DNA editing upstream of a target site. In the reverse prime editing system, a targeted strand nickase (such as D10A-Cas9) is used to cleave a nick on a targeted strand, thereby enabling precise small-fragment DNA base insertion, deletion and substitution upstream of the target site under the action of a reverse transcriptase.

The invention provides a compound for releasing a urine fluorescent biomarker under induction of mitochondrial DNA mutation as well as a preparation method and application of the compound, and belongs to the technical field of medicines. The compound disclosed by the invention is prepared by the following steps: complementarily pairing a hybrid nucleic acid nano-carrier containing single-stranded DNA (Deoxyribose Nucleic Acid), an annular fluorescent reporter molecule and a Cas12a / crRNA compound through bases of DNA or RNA, and then coating polyethyleneimine and hyaluronic acid. A CRISPR / Cas12a system is selected as a converter of tumor specific mitochondrial DNA mutation information and urine fluorescence signals, a nano delivery system which responds to mtDNA single base mutation and is used for tumor progress monitoring and early metastasis warning is designed and synthesized, and the nano delivery system has a good application prospect in tumor progress monitoring and early metastasis sensitive detection.

The present disclosure provides adenosine deaminase, fusion proteins, base editor systems and uses, and specifically provides an adenosine deaminase polypeptide having an amino acid sequence having at least about 70%, 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 13. Also provided are novel nucleic acid modified enzymes having improved efficiency, compositions of base editors comprising the same, and methods and uses for editing target sequences using these adenosine deaminase variants.

The invention belongs to the technical field of biological medicine. The invention relates to a signal peptide, in particular to a signal peptide of GLP-1 and an analogue thereof and application thereof. The invention provides a signal peptide of a GLP-1 protein or an analogue thereof. The amino acid sequence of the signal peptide is shown as any one of SEQ ID NO.1-8. The signal peptide of the GLP-1 protein is improved, so that the signal peptide can promote the generation of an inclusion body and optimize the property of the inclusion body, and also can improve the unit yield of the target protein, and finally, the target protein can obtain more efficient expression. Besides, while the signal peptide is truncated, the base sequence of the target protein is optimized, the character of the inclusion body is changed, and the character of the inclusion body is further optimized. When the fusion protein is used for preparing the GLP-1 protein analogue, the proportion of the target protein in an inclusion body can be greatly improved while the original high inclusion body yield is ensured, the purpose of improving the productivity from the source is finally achieved, and the fusion protein has good application prospects and value.

The invention discloses a method and application for single nucleotide polymorphism identification based on crRNA spacer splitting, by splitting the crRNA spacer complementary to the target base to obtain a recognition probe, when the recognition probe is fully complementary to the target sequence, Cas12a protein is activated to produce a significant fluorescent signal; when there is a base mismatched with the split spacer in the target sequence, the crRNA spacer of the split is difficult to assemble with the incomplete matching sequence, and the Cas12 system is not activated and does not produce a fluorescent signal. The method of the present invention enhances the high specificity recognition ability of CRISPR / Cas12a to single base mismatch, so that the detection of SNP is not limited by the position of the mismatched base, and still shows high resolution in low mutation abundance, for preparing a detection kit for single nucleotide mismatch, to meet the actual detection requirements for clinical samples.

The invention belongs to the technical field of molecular biology, and particularly relates to a DNA binding site of a fungus transcription factor AniJ and application. According to the invention, an echinocandin B gene cluster regulatory factor AniJ is taken as a research object, a ptH-SpRY-ACBE base editor and an sgRNA library covering an aniA promoter are utilized, 5-FOA screening is combined, and a binding motif RNGCTGAS of the AniJ in a core promoter region is excavated. The construction of an engineering strain containing a modified promoter (increasing the number of motifs) proves that the motifs can significantly enhance the expression of aniA and the yield of echinocandin B. And a novel molecular tool is provided for metabolic engineering of natural products of fungi.

The disclosure provides, for example, double stranded DNA (dsDNA) molecules comprising one or more chemically modified nucleobases. In some embodiments, the dsDNA molecule is circular and comprises a first strand and a second strand, wherein the first strand comprises one or more chemically modified nucleobases, and the second strand is free of chemically modified nucleobases. In some embodiments, the dsDNA molecule comprises a promoter sequence and an effector sequence that encodes an effector.

The invention relates to a T7RNA polymerase-guided bacillus subtilis cytosine base editor and application thereof, and belongs to the technical field of gene editing. A T7RNAP guided cytosine base editor is developed in bacillus subtilis, four kinds of cytidine deaminase from different sources are selected to be fused with T7RNAP, the cytidine deaminase is fused at the N end of the T7RNAP, and a fusion body with the highest mutation activity is obtained through screening; the cytosine base editor is used for evolving a global transcriptional regulatory factor CodY, in-situ evolution of genome protein is achieved, and the expression level of bovine-derived beta-lactoglobulin (beta-Lg) is improved. According to the cytosine base editor, the operable range of the cytosine base editor on a genome is widened, multi-site base (C-T) editing can be realized, and an editing window is large.

The invention discloses a high-efficiency wide-targeting-range C-to-G double-enzyme synergistic base editor and application thereof, and belongs to the technical field of gene editing. The editor is a fusion protein comprising a Cas protein having reduced or lost endonuclease activity, a cytosine deaminase (CDA), and a cytosine DNA glycosylase (CDG). In order to solve the problems that an existing C-to-G editor is low in efficiency and limited in targeting range, the C-to-G editor synergistically and efficiently generates a base removal (AP) site on a target DNA through the dual effects of CDA and CDG, so that C-to-G base transversion is promoted. According to the editor, the C-to-G editing efficiency is remarkably improved, an editing window can be effectively expanded or moved, the targeting flexibility is greatly enhanced, meanwhile, high genome specificity is kept, and the editor shows strong application potential in various organisms such as yeast and plants.

Provided is an efficient and precise plant gene knockout method. First, a sequence containing a stop codon cluster is inserted into a genome by means of dual pegRNA to obtain, by means of screening, a stop codon cluster sequence capable of efficiently and precisely knocking out a target gene. A prime editing system and multiple pegRNA programming can be used in combination with the method to enable various genomic modifications such as efficient and precise knockout of one or more target genes, base substitutions, and small fragment insertions, deletions, and substitutions with just one prime editing protein.

The invention belongs to the field of biological medicine, and relates to a guide agRNA for regulating RNA splicing. The invention provides an application of a guide agRNA (Ribonucleic Acid) in preparation of a medicine for changing expression of a target protein by cells of a subject. The guide agRNA recruits ADAR protein to the 3 'splice site or pseudo 3' splice site to edit the A base of the 3 'splice site or pseudo 3' splice site, thereby splicing the whole exon located at the 3 'splice site or flanking the pseudo 3' splice site of the intron from the precursor mRNA, thereby changing the level of mRNA encoding the target protein, and altering the expression of the target protein in the cell. The target gene pre-mRNA selective splicing is regulated and controlled to influence the function or expression of the functional RNA and the target protein of the target gene, so that the purpose of treating diseases is achieved.

The application discloses cytosine deaminase and related biological materials and applications thereof, and belongs to the technical field of proteins. The technical problem to be solved by the application is to mine natural sequence-unpreferred cytosine deaminase, and to improve base editing efficiency and range. The application provides a protein with amino acid sequence of SEQ ID NO. 2 at positions 15-143. The application also provides application of the above protein and related biological materials thereof in single base editing. The application uses macro-genome big data, performs comprehensive and systematic bioinformatics mining analysis on novel cytosine deaminase, and selects a protein L85 highly similar to known DddA protein structure but with low sequence similarity for function verification. The protein L85 has high catalytic activity as cytosine deaminase, and has no target sequence preference restriction, and can be widely applied to in-vitro and in-cell / tissue gene editing, and has good application potential and development value.

The invention relates to an efficient guiding editing system for salvia miltiorrhiza and application of the efficient guiding editing system, and belongs to the technical field of gene editing. The invention provides a guided editing system suitable for salvia miltiorrhiza. The guided editing system comprises a U6complex promoter, a fusion protein and gRNA (guide Ribonucleic Acid), the fusion protein is a PEmax-MLH fusion protein; the nucleotide sequence for compiling the PEmax-MLH fusion protein is as shown in SEQ ID NO. 1; and the gRNA is pegRNA and sgRNA (small guide RNA). The constructed guided editing system realizes diversified and accurate editing in stable transformation of plants, the single-base replacement efficiency reaches 61.0%, the complex insertion replacement efficiency reaches 2.2%, the six-base replacement efficiency reaches 11.6%, and meanwhile, experiments prove that after the guided editing system is adopted to edit salvia miltiorrhiza, the accuracy of the edited salvia miltiorrhiza is greatly improved. The content of tanshinone and salvianolic acid of the edited plant is obviously improved.

The invention discloses a whole genome methylation detection method based on MeDIP-MSRE, and belongs to the technical field of epigenetics detection. According to the method, methylation immunoprecipitation sequencing and a methylation sensitive restriction enzyme technology are innovatively combined, firstly, a methylation specific antibody is used for conducting immunoprecipitation on sample DNA, and whole genome methylation fragments are enriched; then carrying out enzyme digestion on the enriched product by adopting methylation sensitive restriction enzyme, specifically removing an unmethylated DNA region, and reserving a complete methylation sequence; and finally, constructing a methylation map through high-throughput sequencing. According to the method, traditional hydrosulfite chemical conversion is not needed, DNA damage and base conversion deviation caused by the traditional hydrosulfite chemical conversion are avoided, meanwhile, high sensitivity of MeDIP and high specificity of methylation sensitive restriction endonuclease are fused, and the fidelity, sensitivity and specificity of detection are remarkably improved.

The objective of this invention is to provide: aerobic-tolerant Bifidobacterium spp. capable of being cultured under aerobic conditions, a method for culturing the same and a method for screening it, and a composition comprising aerobic-tolerant Bifidobacterium spp. and a method for manufacturing the same. The present invention that solves this problem is a method for culturing Bifidobacterium bacteria, comprising the step of culturing Bifidobacterium bacteria having any one of the following (1) to (7) under aerobic conditions: (1) a gene consisting of a base sequence shown in sequence number 1 or 2; (2) a gene having more than 90% identity with a base sequence shown in sequence number 1 or 2; (3) a gene consisting of a base sequence in which 1 to several bases are missing, substituted, or added to a base sequence shown in sequence number 1 or 2; (4) a gene consisting of a base sequence of DNA that can hybridize under strict conditions with a DNA consisting of a complementary sequence of a base sequence shown in sequence number 1 or 2; (5) a gene consisting of a degenerate isomer of a base sequence shown in sequence number 1 or 2; (6) a gene encoding a protein consisting of an amino acid sequence shown in sequence number 3 or 4; (7) a gene encoding a protein consisting of an amino acid sequence in which 1 to several amino acids are missing, substituted, or added to an amino acid sequence shown in sequence number 3 or 4.